T   OF 


A  COMPARISON  OF  THE  LIFE   CYCLE 
OF  CEITHIDIA  WITH  THAT  OF 
TRYPANOSOMA  IN  THE  IN- 
VERTEBRATE HOST 


A  THESIS  ACCEPTED  IN  PAETIAL  SATISFACTION  OF 
THE  REQUIREMENTS  FOR  THE  DEGREE  OF 

DOCTOR  OF  PHILOSOPHY 
AT  THE  UNIVERSITY  OF  CALIFORNIA 


BY 


IRENE  AGNES  McCULLOCH 


1919- 


A  Comparison   of   the  Morphology   and.   the   Life 
Cycle   of    the   Genus    Gri  thidia  with    that   of 
the   crithidial    stages   of   Tryanosoma. 


Irene  Agnes   Lie  Cul  loch,    A.    B.  ,    L.    A. 
Un  i  v  e  r  s  i  ty  of  Y.  an  s  as  ,    1  9  1  o  . 

A   thesis    submitted    in   partial    fulfillment,  c 

.s    for   the    degree   of  Doctor  of  Philosophy 


-Committee : 


UNIVERSITY    OF    CALIFORNIA    PUBLICATIONS 

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ZOOLOGY 

Vol.  19,  No.  4,  pp.  135-190,  plates  2-6,  3  figures  in  text  October  4,  1919 


A    COMPARISON    OF   THE    LIFE    CYCLE    OF 

CRITHIDIA  WITH  THAT  OF  TRYPANOSOMA 

IN  THE  INVERTEBRATE  HOST 


BY 

IRENE   McCULLOCH 


UNIVERSITY  OF  CALIFORNIA  PRESS 
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UNIVERSITY    OF    CALIFORNIA     PUBLICATIONS 

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ZOOLOGY 

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A  COMPARISON  OF  THE  LIFE   CYCLE   OF 

CRITHIDIA  WITH  THAT  OF  TRYPANO- 

SOMA  IN  THE  INVERTEBRATE 

HOST 

BY 

IRENE  McCULLOCH 


CONTENTS 

PAGE 

Introduction 136 

Historical  summary 139 

The    comparative    morphology    of    Crithidia    and    the    crithidial    stages    of 

Trypanosoma 144 

Nucleus 146 

Blepharoplast 147 

Parabasal  body  and  rhizoplasts 149 

Flagellum  and  undulating  membrane 151 

The  life  cycle  of  Crithidia  euryophthalmi 152 

The  developmental  series 153 

Stomach  phase 153 

Extracellular  crithidias 154 

Oval  spores 154 

Developing  crithidias 154 

Multiple  fission 155 

Endogenous  budding 155 

Somatella 162 

Binary  fission 165 

Intracellular  crithidias  168 

Rectal  phase 170 

Nectomonads 171 

Haptomonads 174 

Final  spore  forms 176 

The  degenerative  series 177 

Conclusions 178 

Literature  cited 180 

Explanation  of  plates 182 


136  University  of  California  Publications  in  Zoology       [VOL.  19 


INTRODUCTION 

In  my  work  on  the  flagellate  parasites  of  hemipteran  insects  during 
the  past  five  years  the  evidence  indicating  a  close  relationship  between 
Crithidia  and  the  crithidial  stages  of  Trypanosoma  has  been  continu- 
ously accumulating  and  has  become  more  and  more  convincing.  The 
relationship  is  shown  both  in  their  morphology  and  in  the  stages  of 
their  life  cycles.  This  paper  presents  a  comparison  of  the  morphology 
and  the  life  cycle  of  the  genus  Crithidia  with  that  of  Trypanosoma  in 
its  crithidial  stages. 

Before  taking  up  this  comparison  a  brief  discussion  of  the  phylo- 
genetic  relations  of  Leptomonas,  Herpetomonas,  Crithidia,  and  Try- 
panosoma will  be  of  value  in  clarifying  the  subject.  In  addition  a 
short  discussion  concerning  the  hosts,  their  food,  and  their  methods  of 
infection  will  give  a  fundamental  conception  of  the  problem  in  hand. 

The  phylogenetic  relation  of  these  intestinal  flagellates,  Lepto- 
monas, Ilerpetomonas,  and  Crithidia,  to  the  haemoflagellates,  or  Try- 
panosoma, has  received  the  attention  of  many  investigators  and 
consequently  has  been  the  subject  of  much  controversy.  Only  a  brief 
statement  of  the  historical  side  of  this  controversy  need  be  given  here. 
Minchin  (1908)  discusses  the  possible  sources  from  which  the  trypano- 
somes  may  have  been  evolved,  namely,  from  the  herpetomonad-like 
and  the  trypanoplasma-like  ancestors.  At  that  time  as  well  as  now 
the  evidence  pointed  to  the  crithidial  (herpetomonad-like)  forms  as 
the  true  recapitulative,  developmental,  primitive  stage  in  the  life 
life  cycle  of  Trypanosoma.  Minchin  (1912)  stated: 

The  types  denoted  by  the  generic  names,  Leptomonas,  Crithidia  and  Try- 
panosoma form  a  perfect  evolutionary  series  with  monogenetic  parasites  of  inver- 
tebrates culminating  in  digenetic  blood  parasites.  It  must  be  emphasized,  however, 
that  any  such  conclusions  are  of  a  tentative  nature  and  can  have  no  finality  but 
are  liable  to  modification  with  every  increase  of  knowledge  concerning  these 
organisms. 

Wenyon  (1913)  also  discusses  the  whole  question  of  the  phylo- 
genetic relationship  of  Leptomonas,  Herpetomonas,  Crithidia  and 
Trypanosoma.  If  the  trypanosome  be  regarded  as  the  highest  stage 
of  development  then  his  conclusion  is  that  the  phylogenetic  order  of 
these  flagellates  would  be :  Leptomonas,  Crithidia,  Herpetomonas,  and 
Trypanosoma.  He  makes  a  distinction  between  the  genera  Lepto- 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma        137 

monas  and  Herpetomonas.  Leptomonas  is  a  flagellate  having  the  non- 
flagellated  and  herpetomonad  form  in  its  life  cycle ;  Herpetomonas 
has  these  two  stages,  together  with  a  crithidial  and  a  trypaniform 
stage.  But  such  a  distinction  has  not  been  generally  accepted.  His 
chief  reason  for  placing  Herpetomonas  next  to  Trypanosoma  is  the 
presence  of  a  trypaniform  phase  in  the  life  cycle  of  a  few  herpeto- 
monad flagellates,  as,  for  example,  those  found  in  Musca  domestica 
and  Drosophila  confusa  (Chatton  and  Leger,  1911). 

From  the  viewpoint  of  the  present  study  of  these  flagellates  the 
hemipteran  insect  hosts  have  been  frequently  divided,  for  convenience, 
into  the  plant-feeding  and  the  blood-sucking  types.  According  to 
Patton  and  Cragg  (1913)  some  species  of  the  three  families  of  plant- 
feeding  Hemiptera,  the  Pentatomidae,  the  Lygaeidae,  and  the  Coreidae 
have  been  found  to  serve  as  hosts  for  either  crithidial  or  herpetomonad 
flagellates.  One  more  family  should  now  be  added  to  this  list,  namely, 
the  Pyrrhocoridae,  to  which  the  " lupine  bug"  (Euryophthalmus  con- 
vivus)  belongs.  Among  the  blood-sucking  Hemiptera  some  species  of 
the  family  Reduviidae  and  the  family  Cimicidae  are  parasitized  by 
haemoflagellates  having  a  cycle  in  a  vertebrate  host  and  in  some  cases 
by  other  flagellates  having  only  the  invertebrate  or  insect  host. 
Among  the  hosts  discovered  in  the  family  Reduviidae  we  find  Tria- 
toma  (==  Conorhinus)  megista  Burm.,  the  invertebrate  host  of  Schizo- 
trypanum  cruzi  (Chagas,  1909)  and  Triatoma  protracta  Uhler,  the 
invertebrate  host  for  Trypanosoma  triatomae  (Kofoid  and  McCulloch, 
1916). 

In  addition  to  the  hemipteran  insects  the  Diptera  (flies,  sheep 
ticks,  Siphonaptera  (fleas),  and  Anapleura  (lice)  also  serve  as  hosts 
for  some  of  the  herpetomonad,  crithidial,  and  trypaniform  flagellates, 
the  rat-flea  (Ceratophyllus  fasciatus)  is  one  of  the  invertebrate  hosts 
of  Trypanosoma  lewisi.  Frequent  reference  will  be  made  to  this 
flagellate  in  the  comparison  of  Crithidia  with  Trypanosoma. 

The  methods  of  infection  of  the  above  hosts  have  been  described 
as  casual,  "cross,"  and  hereditary.  Of  these  three  methods  Patton 
(1908)  proved  that  nymphs  of  Lygaeus  militaris  became  infected  by 
ingesting  with  their  food  the  feces  of  infected  adults.  The  feces  con- 
tained encysted  or  spore  forms.  Porter  (1910)  described  two  addi- 
tional methods  for  the  infection  of  sheep  ticks,  the  "cross,"  and  the 
hereditary.  The  former  occurs  among  insects  with  cannibalistic 
habits,  the  contents  and  parasites  of  the  digestive  tract  of  one  host 
individual  being  eaten  by  another.  The  hereditary  infection  has  been 


140  University  of  California  Publications  in  Zoology        [VOL.  19 

pathogenic  to  its  vertebrate  host,  the  rat.  The  invertebrate  host  of 
this  haemoflagellate  is  normally  the  rat-flea,  which  transmits  the 
flagellate  from  rat  to  rat.  Both  the  vertebrate  and  the  invertebrate 
host  of  T.  lewisi  are  widespread,  abundant,  easily  procurable,  and 
adaptable  to  laboratory  conditions.  To  make  certain  that  none  of  the 
stages  of  the  life  history  of  the  so-called  natural  flagellates  should 
be  confused  with  stages  of  the  life  history  of  T.  lewisi  a  stock  of  un- 
infected  fleas  was  procured  for  the  breeding  cages.  The  rat-fleas  have 
frequently  been  found  to  be  infected  with  Leptomonas  pattoni. 

At  the  time  of  the  publication  of  the  Minchin  and  Thomson  paper 
I  was  working  on  the  morphology  and  life  history  of  Crithidia  eury- 
ophthalmi  (McCulloch,  1917),  a  form  which  occurs  in  the  alimentary 
tract  of  Euryophthalmus  convivus.  This  material  was  of  great  in- 
terest; the  intracellular  process  of  multiple  fission  was  found,  and 
the  initial  infective  spores  were  relatively  abundant.  Previously 
some  time  had  been  spent  in  studying  the  morphology  and  life  history 
of  Crithidia  leptocoridis  (McCulloch,  1915),  which  infects  the  digestive 
tract  of  the  box  elder  bug,  Leptocoris  trivittatus.  It  had  also  been 
pointed  out  in  a  superficial  way  that  the  individuals  of  various  forms, 
shapes,  and  structures  found  in  a  typical  infection  of  this  crithidia, 
C.  leptocoridis,  were  apparently  analogous  to  many  of  the  correspond- 
ing figures  of  Schizotrypanum  cruzi  (Chagas,  1909)  in  the  invertebrate 
host.  As  the  investigation  of  C.  euryophthalmi  and  C.  leptocoridis 
proceeded  it  became  increasingly  easy  to  link  the  life  history  of 
Critkidia  with  the  life  history  of  Trypanosoma  in  the  invertebrate 
host.  This  was  especially  true  of  the  life  history  of  T.  lewisi.  In 
order  to  demonstrate  clearly  the  essential  facts  of  the  life  cycle  of  one 
of  these  important  flagellates  a  brief  outline  of  the  life  history  of 
T.  lewisi  will  be  given,  based  upon  the  work  of  Minchin  and  Thomson. 

The  developmental  cycle  of  T.  lewisi  in  the  flea  is  divided  into  two 
phases,  characteristic  of  the  parts  of  the  digestive  tract  in  which  the 
trypanosomes  are  found,  viz.,  the  stomach  and  the  rectal  phase.  The 
cycle  in  the  rat-flea  requires  a  minimum  of  five  days  for  its  complete 
course.  The  trypanosomes  enter  the  stomach  of  the  flea  with  the 
blood  of  an  infected  rat.  They  show  the  characteristic  structure  of  a 
trypanosome,  the  "  kinetonucleus, "  or  parabasal  body,  being  posterior 
to  the  nucleus  and  the  undulating  membrane  well  developed.  The 
change  in  the  medium  is  probably  responsible  for  the  physiological 
changes  which  result  in  the  bodies  becoming  more  cylindrical.  The 
trypanosomes  then  penetrate  the  epithelial  cells  of  the  stomach  and 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma        141 

undergo  the  process  of  multiple  fission,  a  process  which  characterizes 
the  stomach  phase.  The  initial  steps  in  this  process  after  the  trypano- 
some  has  entered  an  epithelial  cell  of  the  stomach,  is  the  formation 
of  a  "tailed"  sphere.  This  is  a  spherical  structure  with  the  flagellum 
and  some  cytoplasm  protruding  at  one  point.  A  little  later  the 
the  ' '  tailed ' '  sphere  becomes  ' '  tailless, ' '  and  shows  internally  the  form- 
ation of  a  variable  number  of  daughter  individuals.  In  the  forma- 
tion of  these  daughter  individuals,  the  nucleus,  parabasal  body,  and 
blepharoplast  each  divide  an  equal  number  of  times,  forming  mero- 
zoites  of  a  crithidiomorphic  type,  i.e.,  a  long,  free  type  of  trypano- 
some  whose  external  movements  are  crithidial  but  whose  structure  still 
shows  the  parabasal  body  posterior  to  the  nucleus.  The  crithidio- 
morphic flagellates  may  do  one  of  two  things:  enter  other  epithelial 
cells  and  undergo  the  process  of  multiple  fission,  or  collect  at  the 
pyloric  opening  of  the  stomach,  to  be  carried  down  through  the  intes- 
tine into  the  rectum  with  the  food.  The  intestine  ordinarily  serves  as 
a  passageway  for  the  parasites  from  the  stomach  to  the  rectum,  but 
under  certain  conditions  rectal  forms  may  migrate  forward  and  attach 
themselves  to  the  wall  of  the  intestine  in  the  postpyloric  region. 

The  rectal  phase  is  established  by  the  entrance  of  the  crithidio- 
morphic forms  into  this  region  of  the  digestive  tract,  and  this  region 
becomes  the  permanent  source  of  infection  throughout  the  life  of  the 
flea.  During  the  migration  from  the  stomach  to  the  rectum  structural 
changes  take  place  in  the  crithidiomorphic  trypanosomes.  The  poste- 
rior end  of  the  body  becomes  club-shaped,  and  this  shifting  of  the 
cytoplasm  assists  in  the  forward  movement  of  the  parabasal  body  to 
a  position  anterior  to  the  nucleus.  Following  this  condition,  binary 
fission  brings  about  the  production  of  the  smaller,  minute  crithidias 
which  characterize  this  phase  of  the  life  history.  The  established  rectal 
phase  is  described  as  consisting  of:  (1)  the  attached  or  haptomonad 
form,  which  is  the  multiplicative  stage  of  the  rectal  development; 
(2)  the  free  or  nectomonad  form;  and  (3)  the  final  trypanosome  form 
which  is  instrumental  in  infecting  another  host. 

The  above  outline  of  the  life  history  deals  only  with  the  develop- 
mental series;  but  in  both  the  stomach  and  rectum  there  are  present 
individuals  which  show  degeneration  and  belong  consequently  to  the 
degenerative  series.  I  have  found  that  the  life  history  of  Crithidia 
euryophthalmi  can  be  correlated  advantageously  with  the  life  his- 
tory of  T.  lewisi  in  the  invertebrate  host,  the  flea.  The  life  history 
of  C.  euryophthalmi  is  not  the  life  history  of  a  haemoflagellate  but 


142  University  of  California  Publications  in  Zoology       [VOL.  19 

apparently  that  of  a  more  primitive  flagellate  (pis.  2-6)  found  living 
in  the  medium  of  the  fluid  contents  of  the  alimentary .  tract  of  a 
plant-feeding  insect.  As  such,  C.  euryophthalmi  (fig.  A,  2)  lacks  in 
the  stomach  phase  the  trypaniform  characteristics,  namely,  the  ble- 
pharoplast  (bl.),  the  parabasal  (pi).)  (or  kinetonucleus)  located  poste- 
rior to  the  nucleus  (n.),  and  the  well  developed  undulating  membrane. 
In  this  connection  it  is  interesting  to  note  that,  regardless  of  the  con- 
trast in  the  initial  stages  of  the  stomach  phase  in  each  life  cycle,  the 
process  of  multiple  fission  characterizes  the  stomach  phase  for  both 
the  haemoflagellate  and  the  more  primitive  crithidial  flagellate.  In 
the  stomach  phase  of  the  crithidial  parasite  there  are  also  found  the 
"rounding  up"  forms  (pi.  3,  figs.  24,  25),  the  spheres  (pi.  3,  figs. 
26-32),  and  the  final  stages  of  multiple  fission  wherein  the  resulting 
merozoites  are  about  to  escape  from  the  epithelial  cell  of  the  host  (pi.  3, 
fig.  39) .  In  addition  to  this  I  found  numerous  stages  of  an  endogenous 
process  of  multiple  fission  (pi.  2,  figs.  11-23). 

Correlation  of  the  life  histories  of  these  forms  (C.  euryophthalmi 
and  T.  lewisi)  became  more  difficult  in  the  stage  following  the  process 
of  multiple  fission,  on  account  of  the  structure  of  the  digestive  tract 
of  the  host,  Euryophthalmus  convivus.  The  digestive  tract  of  the  flea 
(cf.  Minchin  and  Thomson,  1915,  text-fig.  1)  shows  the  stomach  as  a 
prominent  enlargement  of  the  tube,  followed  by  a  comparatively  long, 
slender  intestine,  at  the  posterior  end  of  which  is  the  rectal  enlarge- 
ment. The  digestive  tract  of  Euryophthalmus  convivus,  or  lupine  bug, 
on  the  other  hand,  is  quite  unlike  that  of  the  flea  (cf.  McCulloch,  1917, 
text-fig.  1 ) .  The  first  enlargement  of  the  mid-gut  proper  is  followed  by 
a  second  and  a  third  enlargement,  a  narrow  constriction  of  the  diges- 
tive tract  separating  the  three  expansions.  Posterior  to  these  there  is  a 
relatively  long  intestine  which  passes  through  the  center  of  a  ruffled, 
ribbon-like  gland.  The  intestine  opens  into  the  slight  enlargement 
near  the  entrance  of  the  malpighian  tubules,  which  in  turn  opens  into 
the  rectum.  Since  the  several  parts  of  the  digestive  tract  of  the  hem- 
ipteran  and  other  insects  have  not  been  satisfactorily  homologized  as 
yet  and  the  nomenclature  used  in  describing  the  divisions  is  confused, 
it  is  exceedingly  difficult  to  ascertain  the  homology  of  these  several 
parts  of  the  digestive  tract.  However,  as  indicated  in  my  preliminary 
paper  (McCulloch,  1917),  all  three  enlargements  anterior  to  the  intes- 
tine were  considered  as  parts  of  the  stomach  proper  and  were  accord- 
ingly designated  as  the  "crop,"  mid-stomach,  and  pyloric  expansion. 
With  this  disposal  of  the  three  enlargements  of  the  digestive  tract,  the 


1919J      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma        143 

question  immediately  arose  as  to  whether  or  not  all  the  crithidial  infec- 
tions of  these  regions  are  a  part  of  the  stomach  phase  of  the  life 
history  of  C.  euryophthalmi.  A  careful  study  of  the  crithidial  infec- 
tion of  each  of  these  portions  of  the  digestive  tract  together  with  many 
examinations  of  the  contents  of  the  intestine,  the  gland,  and  the 
rectum,  convinced  me  that  food  conditions  do  not  permit  the  parasites 
to  establish  a  permanent  rectal  phase  in  the  rectum  but  that  they 
are  forced  to  remain  anterior  to  the  intestine,  in  the  pyloric  expansion. 
Preparations  of  the  gland  and  intestine  have  as  yet  shown  no  infection 
posteriorly,  and  the  rectum  has  contained  an  occasional  infection  of 
spore  forms  only. 

To  add  to  the  difficulty  in  determining  the  extent  of  the  stomach 
phase  and  the  beginning  of  the  rectal  phase  no  structural  changes  of  a 
striking  nature  occur  in  the  stomach  phase  of  C.  euryophthalmi  as  is 
the  case  in  T.  lewisi;  nevertheless,  the  behavior  of  the  crithidias  is  of 
some  assistance  and  the  study  of  the  serial  sections  and  stained  smears 
of  the  several  parts  have  added  materially  to  our  knowledge  of  the 
phases.  Taking  into  consideration  the  evidence  from  these  sources 
the  crithidial  infection  of  the  "crop"  is  interpreted  as  the  stomach 
phase.  The  mid-stomach  serves  as  a  temporary  location  for  the  slowly 
migrating  forms  of  the  stomach  phase,  and  the  pyloric  expansion  be- 
comes the  region  of  the  permanent  location  of  the  "rectal"  phase 
during  the  life  of  the  lupine  bug.  The  established  "rectal"  phase 
of  the  life  history,  of  C.  euryophthalmi  in  the  pyloric  expansion  has 
three  general  types  of  individuals,  the  attached,  or  haptomonad,  the 
free,  or  nectomonad,  and  the  final  spore  forms,  which  probably  serve 
to  infect  another  insect.  Only  the  last  type  of  parasite  has  been  found 
in  the  normal  contents  of  the  rectum. 

With  these  brief  explanatory  outlines  of  the  life  histories  of  T. 
lewisi  and  of  C.  euryophthalmi  a  detailed  discussion  of  the  more  im- 
portant points  concerning  the  comparative  morphology  of  the  crith- 
idial stages  of  Trypanosoma  and  of  Crithidia  will  now  be  given.  The 
comparison  of  the  life  cycles  which  follows  is  based  upon  the  work 
of  Minchin  and  Thomson  on  the  life  cycle  of  T.  lewisi,  and  upon 
the  life  history  of  C.  euryophthalmi  with  special  reference  to  the 
accompanying  plates  (pis.  2-6).  For  further  details  of  the  life 
history  of  T.  lewisi  the  reader  is  referred  to  Minchin  and  Thomson's 
paper  (1915). 


144  University  of  California  Publications  in  Zoology        [VOL.  19 


THE    COMPARATIVE    MORPHOLOGY   OP    CRITHIDIA   AND 
THE  CRITHIDIAL  STAGES  OF  TRYPANOSOMA 

The  morphology  of  Crithidia  and  Trypanosoma  has  been  the  sub- 
ject of  careful  investigation  for  a  number  of  years,  and  our  concep- 
tion of  the  structure  of  these  simple  organisms  has  been  modified  from 
time  to  time  by  additional  discoveries.  This  is  especially  true  of 
the  extranuclear  organelles  of  these  flagellates,  owing  to  the  recent 
investigations  carried  on  in  the  Zoological  Laboratory  of  the  Uni- 
versity of  California  by  Dr.  C.  A.  Kofoid  (1916)  and  Dr.  Olive 
Swezy  (1916),  the  latter  studying  particularly  the  binuclear  theory 
of  Hartmann  (1911).  The  work  of  these  investigators  has  centered 
attention  upon  the  extranuclear  organelles  of  these  flagellates,  con- 
sisting of  the  blepharoplast  (fig.  A,  U.)  at  the  base  of  the  flagellum, 
the  parabasal  body  (pb.),  or  kinetonucleus,  the  rhizoplast  (r/«.),  the 
parabasal  rhizoplast  (pb.  rh.),  the  flagellum  (fl.),  and  the  undulating 
membrane  (und.  m.).  They  have  homologized  the  kinetonucleus  of 
the  Protomonadina  (Herpetomonas,  Crithidia,  and  Trypanosoma} 
with  the  parabasal  body  of  the  Polymastigina  and  the  Hypermasti- 
gina.  Bearing  this  in  mind,  it  at  once  becomes  clear  that  this  extra- 
nuclear  complex  of  organelles  is  the  neuromotor  apparatus  of  Crith- 
idia and  Trypanosoma  (Kofoid,  1916).  In  a  previous  paper  (Kofoid 
and  McCulloch,  1916)  the  term  parabasal  body  was  used  in  place  of 
kinetonucleus  throughout  and  I  shall  employ  this  nomenclature  in 
the  present  paper. 

The  position  of  this  extranuclear  complex  of  organelles  determines 
largely  whether  the  flagellate  is  a  trypanosome  or  a  crithidia.  The 
trypanosome  is  characterized  by  the  presence  of  the  parabasal  body 
and  the  blepharoplast  posterior  to  the  nucleus,  and  by  a  well  developed, 
undulating  membrane  which  passes  forward  laterally  along  the  edge 
of  the  ribbon-like  body.  These  characteristics  are  common  to  the 
flagellates  found  in  the  blood,  and  modifications  of  this  structure  take 
place  as  soon  as  the  medium  is  changed,  as  in  the  transfer  to  the 
stomach  of  the  flea.  The  transition  stages  between  a  trypanosome  and 
a  crithidia  have  been  designated  as  crithidiomorphic  trypanosomes  by 
Minchin  and  Thomson,  as  previously  noted.  In  the  transition  forms 
the  parabasal  body  and  the  blepharoplast  are  still  posterior  to  the 
nucleus  at  a  greater  or  less  distance,  but  the  movement  and  shape  of 
the  body  of  the  flagellate  are  distinctly  like  those  of  a  crithidia.  In 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma         145 

the  crithidial  forms  the  extranuclear  complex  of  organelles  is  anterior 
to  the  nucleus  (fig.  A,  1,  2)  and  the  body  has  a  tendency  to  be 
cylindrical  in  cross-section  and  to  show  only  a  slight  undulating  mem- 
brane (und.  m.,  fig.  A).  These  crithidial  forms  are  common  to  the 
life  cycle  of  both  Crithidia  and  Trypanosoma.  The  next  step  in  our 


Fig.  A.  Typical  crithidial  forms  of:  (1)  Crithidia  leptocoridis ;  (2)  C. 
euryophthalmi;  (3)  Trypanosoma  lewisi  (after  Minchin  and  Thomson,  1915,  pi. 
37,  fig.  66);  (4)  T.  triatomae;  (5)  Schizotrypanum  crusi  (after  Chagas,  1909, 
pi.  13,  fig.  16) ;  showing  the  similarity  of  the  several  flagellates  in  size,  form 
and  structure.  X  3500.  1)1.,  blepharoplast;  ft.,  flagellum;  n.,  nucleus;  pb.,  para- 
basal  body;  ph.  rh.,  parabasal  rhizoplast;  n.  rh.,  nuclear  rhizoplast;  und.  m., 
undulating  membrane;  vac.,  vacuole. 

discussion  will  be  directed  to  the  demonstration  of  the  close  resem- 
blance of  the  crithidial  forms  of  the  genus  Crithidia  to  those  of 
Trypanosoma  in  the  invertebrate  host. 

A  series  of  typical  crithidial  flagellates  (fig.  A)  has  been  selected 
from  the  stomach  phase  of  the  life  histories  of  both  Crithidia  and 


146  University  of  California  Publications  in  Zoology        [VOL.  19 

Trypanosoma  to  show  in  detail  the  comparative  morphology  in  this 
particular  stage.  C.  leptocoridis  (fig.  A,  1)  and  C.  euryophthalmi 
(fig.  A,  2)  have  been  selected  to  represent  the  structure  of  Crithidia 
and  Trypanosoma  leivisi  (fig.  A,  3,  after  Minchin  and  Thomson,  1915, 
pi.  37,  fig.  66),  T.  triatomae  (fig.  A,  4),  and  Schizotrypanum  cruzi 
(fig.  A,  5,  after  Chagas,  1909,  pi.  13,  fig.  16)  that  of  Trypanosoma. 

Each  crithidial  flagellate  of  this  series  has  an  elongate  body, 
cylindrical  in  outline  but  slightly  flattened  at  the  anterior  portion, 
which  forms  the  undulating  membrane.  At  the  edge  of  this  membrane 
there  is  a  sharply  defined  flagellum  of  variable  length  (fig.  A,  1,  3). 
The  length  of  the  flagellum  has  no  particular  significance  since  con- 
siderable variation  exists  within  each  species.  Posteriorly,  however, 
there  is  in  the  stomach  phase  a  consistent  difference  between  the 
crithidial  form  of  Crithidia  and  the  crithidial  form  of  Trypanosoma. 
The  posterior  ends  of  the  bodies  of  the  true  crithidias  are  more  atten- 
uate (fig.  A,  1,  2)  in  the  stomach  phase,  and  do  not  show  the  slight 
tendency  to  become  club-shaped  until  the  rectal  phase  is  reached.  In 
figure  A,  4,  T.  triatomae  is  quite  blunt  at  the  posterior  end,  and  the 
shifting  of  the  cytoplasm  into  this  region  is  increasing  the  width  of 
the  body  at  the  expense  of  the  length.  Another  difference  almost  as 
consistent  as  the  one  just  noted  is  the  variation  in  the  nucleus  (n.). 
In  Crithidia  (fig.  A,  1,  2)  it  is  usually  anterior  to  the  center,  and. in 
the  crithidial  forms  of  Trypanosoma  it  is  posterior  to  the  center  (fig. 
A,  3,  4,  5). 

Internally  the  similarity  of  the  structure  of  the  organelles  and  their 
relationship  in  general  is  quite  marked.  The  nucleus  (n.),  the  ble- 
pharoplast  (bl.),  and  the  parabasal  body  (ph.)  are  common  to  each 
of  these  flagellates.  The  nuclear  rhizoplast  (rh.)  and  the  parabasal 
rhizoplast  (pb.  rh.)  are  found  in  all  the  above  flagellates  with  the 
exception  of  T.  lewisi  (fig.  A,  3).  The  absence  of  these  organelles  in 
the  crithidial  form  of  T.  leivisi  is  questionable  since  they  are  present 
in  all  the  others.  They  may  have  been  overlooked  because  of  the 
delicacy  of  their  structure  and  the  faintness  with  which  they  stain. 

The  nucleus  and  the  extranuclear  organelles  will  now  be  taken  up 
in  detail. 

Nucleus. — This  organelle  in  each  of  the  above  flagellates  may  be 
described  as  round  or  slightly  oval  in  shape  and  of  the  vesicular  type. 
It  varies  somewhat  in  size  from  I/A  in  figure  A,  3  to  1.7/*  in  figure 
A,  5,  but  it  is  usually  about  two-thirds  of  the  width  of  the  body  in 
diameter.  In  Crithidia  leptocoridis  the  nucleus  shows  clearly  the  ves- 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma         147 

icular  type  of  structure,  having  a  relatively  large  karyosome,  a  light 
area  outside  of  this,  and  a  clearly  defined  nuclear  membrane.  In 
figure  A,  3  a  slightly  different  condition  exists ;  the  nucleus  of  C.  euryo- 
phthalmi  is  of  the  vesicular  type,  but  the  nuclear  membrane  is  en- 
crusted with  considerable  chromatin,  and  the  central  karyosome  is 
somewhat  reduced  as  compared  with  that  of  C.  leptocoridis  in  figure 
A,  1.  An  examination  of  plate  5  indicates  that  the  nucleus  of  this 
flagellate  (C.  euryophthalmi)  is  characterized  by  the  heavily  encrusted 
nuclear  membrane,  the  small  central  karyosome,  and  the  broad,  clear 
area  between  the  membrane  and  karyosome. 

In  the  trypanosomes  (fig.  A,  3,  4,  5)  the  nucleus  is  slightly  poste- 
rior to  the  center  of  the  body,  which  has  a  tendency  to  be  shorter  and 
more  club-shaped  at  the  posterior  end  than  is  that  of  the  crithidias 
(fig.  A,  1,  2).  The  nucleus  of  Trypanosoma  lewisi  (fig.  A,  3)  shows 
a  peculiar  structure.  In  this  particular  form  it  is  difficult  to  demon- 
strate the  nuclear  membrane,  since  the  light  area  encircling  the  more 
deeply  stained  portion  could  be  interpreted  as  either  intranuclear 
or  extranuclear.  However,  taking  into  consideration  other  figures  of 
T.  lewisi  (cf.  Minchin  and  Thomson,  1915,  pis.  37,  42)  this  light  area 
has  been  regarded  as  being  extranuclear  and  the  diameter  is  I/A 
instead  of  1.4/*.  Within  this  relatively  deep-staining,  nucleus  the 
chromatin  is  divided  unequally  into  a  large,  irregularly  shaped  granule 
and  a  smaller  one.  The  nucleus  of  T.  triatomae  (fig.  A,  4)  resembles 
that  of  C.  euryophthalmi.  It  is  also  characterized  by  the  chromatin- 
encrusted  nuclear  membrane,  the  wide  clear  area,  and  the  small  cen- 
tral karyosome,  as  in  C.  euryophthalmi.  A  nucleus  of  a  somewhat 
more  complex  type  is  found  in  Schizotrypanum  cruzi  (fig.  A,  5).  A 
faint  nuclear  membrane  limits  the  clear  nuclear  space  which  sur- 
rounds the  central  karyosome  containing  a  centriole  at  the  base  of 
the  rhizoplast.  This  nucleus  is  1.7/x  in  diameter  and  somewhat  larger 
than  that  of  any  other  flagellate  in  the  series. 

Blepharoplast. — This  structure  is  of  great  interest  since  it  is  the 
center  of  the  extranuclear  organelles,  or  neuromotor  apparatus.  In 
the  crithidias  this  structure  is  not  a  sharply  defined  basal  granule  at 
the  base  of  the  flagellum.  For  instance,  in  Crithidia  leptocoridis  a 
slight  enlargement  at  the  base  of  the  flagellum  is  noted  (fig.  A,  1). 
Since  this  slight  enlargement  stains  with  almost  the  same  intensity  as 
does  the  flagellum  it  is  exceedingly  difficult  to  get  a  satisfactory  concep- 
tion of  its  structure.  By  careful  focusing  of  the  binocular  microscope 
with  Watson's  no.  20  holoscopic  oculars,  however,  the  small  enlarge- 


148  University  of  California  Publications  in  Zoology       [VOL.  19 

ment  of  slightly  greater  staining  intensity  can  be  observed  at  the 
junction  of  the  parabasal  rhizoplast  with  the  nuclear  rhizoplast.  In 
C.  euryophthalmi  (fig.  A,  2)  even  greater  difficulty  is  experienced  in 
endeavoring  to  find  the  blepharoplast.  In  this  form  there  is  no  en- 
largement of  the  base  of  the  fllagellum,  but  the  area  at  the  junction  of 
the  rhizoplast  is  darker  in  appearance,  and  frequently  a  small  granule 
at  one  side  can  be  seen  by  careful  focusing. 

Among  the  trypanosomes  the  blepharoplast  is  apparently  a  more 
clearly  defined  structure.     Our  investigations  of  T.  triatomae    (fig. 
A,  4)  have  yielded  more  tangible  results.    We  found  in  this  trypano- 
some  a  slight  enlargement  of  the  base  of  the  flagellum  but  no  differ- 
entiation in  the  staining  capacity  of  the  flagellum  and  basal  granule. 
In  T.  lewisi  (fig.  A,  3)  and  in  Schizotrypanum  cruzi  (fig.  A,  5)  the 
figures  of  these  trypanosomes  show  what  is  evidently  a  well  defined 
basal  granule,  or  blepharoplast.    This  is  particularly  true  of  T.  lewisi 
(fig.  A,  3).    In  both  the  crithidias  and  the  trypanosomes  the  blephar- 
oplast frequently  divides  independently  of  the  nucleus.    In  the  binary 
fission  of  C.   euryophthalmi  the  blepharoplast   and  parabasal  body 
may  divide  before  the  nucleus  divides  (pi.  3,  fig.  4),  or  the  nucleus  may 
divide  first  (pi.  3,  fig.  33).    In  figure  25,  plate  3,  the  nucleus  is  divid- 
ing but  there  are  no  indications  of  the  division  of  blepharoplast  and  of 
the  parabasal  body.     A  similar  condition  exists  among  the  trypano- 
somes, indicating  that  the  blepharoplast  is  the  kinetic  center  of  the 
extranuclear  organelles,  or  neuromotor  apparatus..     But  this  is  not 
the  only  kinetic  center  of  these  simple  organisms.     The  nucleus  also 
contains  a  kinetic  center  which  initiates  division  in  the  form  of  a 
centrosome,  at  the  base  of  the  nuclear  rhizoplast  in  those  flagellates 
containing  this  organelle.    In  the  nucleus  of  S.  cruzi  (fig.  A,  5)  this 
centriole,  or  centrosome,  at  the  base  of  the  nuclear  rhizoplast  is  clearly 
defined  within  the  central  karyosome  of  the  nucleus.     The  origin  of 
the  blepharoplast  is  still  the  subject  of  investigation  and  as  yet  110 
conclusive  evidence  is  at  hand  to  establish  beyond  doub't  the  way  in 
which  this  structure  originates.     In  the  endogenous  buds,  which  are 
exceedingly  small  and  difficult  to  interpret,  the  blepharoplast  seem- 
ingly originates  from  the  single  nuclear  structure  as  an  outgrowth 
rather  than  by  a  mitotic  process.     The  outgrowth  forms  a  second 
deep-staining  mass  anterior  to  the  nucleus,   and  at  this  stage  the 
nonflagellated  forms  have  the  appearance  of  "binucleated"  spores. 
As  development  proceeds  the  kinetic  center  of  the  blepharoplast  is 
probably  established  as  the  center  of  the  neuromotor  apparatus,  and 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma        149 

from  this  blepharoplast  the  flagellum  grows  anteriorly  and  the  para- 
basal  body  to  one  side.  Connecting  the  parabasal  body  with  the 
centrosome,  or  blepharoplast,  is  the  parabasal  rhizoplast  (p~b.  rh.). 
Parallel  cases  indicating  such  an  origin  of  the  blepharoplast  can  be 
found  among  other  flagellates. 

Parabasal  body  and  rhizoplasts. — In  all  of  these  flagellates  the 
parabasal  body  (pb.)  presents  the  same  general  appearance  with 
respect  to  its  location,  size  and  shape.  In  C.  leptocoridis  (fig.  A,  1) 
the  parabasal  body  is  a  bar-shaped  structure  made  up  of  two  deeply 
stained  granules  lying  in  close  proximity.  Immediately  surrounding 
this  deep-staining  bar  is  a  clear  area,  limited  by  a  sacklike  covering 
of  dense  cytoplasm,  evidently  continuous  with  the  contractile  cyto- 
plasmic  sheath  of  the  flagellum.  Between  the  parabasal  body  and  the 
blepharoplast  region  the  cytoplasmic  sheath  takes  on  the  appearance 
of  a  cone-shaped  mass  of  contractile  fibers  (p~b.  rh.),  the  apex  of 
which  is  found  at  the  base  of  the  flagellum  and  continuous  with  the 
outer  contractile  cytoplasm  of  the  flagellum.  The  axial  or  central 
part  of  this  parabasal  rhizoplast  is  presumably  of  blepharoplastic 
origin  and  connects  the  blepharoplast  and  parabasal  body. 

The  parabasal  body  of  C.  euryopkthalmi  is  similar  to  that  of 
C.  leptocoridis.  It  is  not  so  large  comparatively  (fig.  A,  2)  and  it 
seldom  has  the  bilobed  appearance.  In  C.  euryophthalmi  the  most 
interesting  and  valuable  evidence  has  been  found  concerning  the 
relation  of  the  parabasal  body  to  the  blepharoplast.  In  text  figure 
B,  1-5  a  series  of  flagellates  has  been  drawn  from  good  iron-haema- 
toxylin  preparations  to  show  clearly  that  the  position  of  the  organelle 
is  lateral  and  not  axial.  In  figure  B,  1  the  end  of  the  bar-shaped 
parabasal  body,  which  lies  lateral  to  the  nuclear  rhizoplast,  extends 
outward  to  the  periplast.  From  another  angle  the  entire  length  of 
this  organelle  is  visible  (fig.  B,  2)  peripheral  to  the  nuclear  rhizoplast, 
which  passes  behind  and  underneath  the  parabasal  rhizoplast.  Still 
another  aspect  of  the  possible  position  of  this  structure  is  indicated 
in  figures  B,  3,  4.  In  each  of  these  figures  it  is  medial  to  the  nuclear 
rhizoplast  and  behind  or  beneath  it  as  it  passes  from  the  centro- 
some of  the  nucleus  to  the  centrosome,  or  blepharoplast,  at  the  base  of 
the  flagellum.  In  such  a  figure  as  B,  5  the  relationship  of  this  organ- 
elle to  the  others  of  the  neuromotor  apparatus  can  be  seen  most  clearly. 
The  cytoplasm  has  become  exceedingly  clear  and  does  not  conceal  the 
other  structure.  The  parabasal  body  in  the  flagellate  stains  very 
deeply  and  has  the  appearance  of  a  chromotoidal  mass  suspended  in 


150  University  of  California  Publications  in  Zoology       [VOL.  1! 

a  sacklike  structure  from  the  region  of  the  blepharoplast.  Unlike  th< 
suspensory  apparatus  of  C.  leptocoridis ,  C.  euryophthalmi  does  no 
show  a  fan-shaped  mass  of  contractile  cytoplasmic  fibrils  covering  this 
axial  portion,  which  is  of  blepharoplastic  origin  (figs.  A,  1,  2 ;  figs.  B 
1-5).  The  outline  of  this  cytoplasmic  envelope  in  C.  euryophthalmi  i: 
definite,  slightly  opaque,  and  continuous  with  the  cytoplasmic  sheatl 
of  the  flagellum. 

The  parabasal  body  of  the  trypanosomes  (figs.  A,  3,  4,  5)  is  simi 
larly  a  bar-shaped  structure,  which  stains  deeply,  located  to  on< 
side  of  the  nuclear  rhizoplast  and  blepharoplast.  In  T.  leivisi  (fig 
A,  3)  the  parabasal  body  is  relatively  small  while  the  blepharoplast  ii 
correspondingly  large.  As  previously  noted,  no  parabasal  rhizoplas 
has  been  figured  by  Minchin  and  Thomson  in  T.  Lewisi.  Here  am 
there  suggestions  of  a  connection  might  be  pointed  out  in  their  figures 
While  it  is  possible  that  T.  lewisi  is  exceptional  in  this  respect,  yet 
since  the  structure  is  found  in  other  flagellates  of  this  group,  we  ma] 
infer  that  a  critical  study  of  the  preparations  with  a  binocular  micro 
scope  will  reveal  the  presence  of  such  a  connection  in  the  crithidia 
stages  of  this  flagellate. 

Trypanosoma  lewisi  (fig.  A,  3)  is  likewise  the  only  flagellate  her< 
figured  in  the  crithidial  stages  without  a  nuclear  rhizoplast.  Her< 
again  T.  leivisi  is  either  exceptional  or  the  structure  has  perhaps  beer 
overlooked,  since  it  is  usually  discerned  with  difficulty.  Chagas  (1909] 
has  figured  a  nuclear  rhizoplast  in  Schizotrypanum  cruzi  and  we  hav< 
found  it  also  in  T.  triatomae.  Therefore  we  may  expect  that  it  wil 
be  found  in  T.  lewisi. 

The  parabasal  body  in  Trypanosoma  triatomae  (fig.  A)  is  a  rela 
tively  large  structure,  its  width  approximately  equal  to  one-half  of  it! 
length.  It  is  located  a  short  distance  anterior  to  the  nucleus,  and  is  sus 
pended  from  the  blepharoplast  by  a  fan-shaped  parabasal  rhizoplas 
like  that  of  Crithidia  leptocoridis,  i.e.,  a  suspensory  apparatus  with  j 
fibrous  appearance.  In  the  crithidial  stage  of  T.  triatomae  a  nucleai 
rhizoplast  was  observed  connecting  the  blepharoplast  with  the  centriol< 
of  the  karyosome,  but  such  a  connection  was  not  found  in  the  trypani 
form  individuals.  If  a  nuclear  rhizoplast  be  present  in  the  trypaniforn 
stage,  in  which  we  did  not  find  it,  it  is  possible  that  we  may  have  beer 
prevented  from  observing  it  because  of  the  density  of  the  cytoplasn 
in  this  stage  and  a  tendency  in  this  delicate  thread  in  the  trypano 
somes  to  stain  lightly.  In  the  crithidial  stages  of  this  trypanosome,  or 
the  other  hand,  the  cytoplasm  is  more  or  less  vacuolated  in  appear 
ance  thus  making  the  nuclear  rhizoplast  more  evident.  When  a  try 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma        151 

panosome  rounds  up  for  the  process  of  multiple  fission  no  nuclear 
rhizoplast  is  in  evidence,  but  after  the  complete  transition  into  the 
crithidial  form  the  nuclear  rhizoplast  can  be  readily  demonstrated 
with  the  high  power  binocular  microscope  and  Watson's  holoscopic 
eyepiece.  It  is  also  possible  that  further  work  will  show  that  the 
centrosomic  structure  of  the  trypaniform  flagellate  differs  from  that 
of  the  crithidial  form,  and  consequently  that  the  rhizoplasts  are  absent 
in  the  former. 

In  Schizotrypanum  cruzi  (fig.  A,  5)  the  parabasal  body  is  slightly 
bilobed  in  appearance,  and  is  suspended  from  the  blepharoplast  by  a 
clearly  defined,  fibrous  rhizoplast.  The  suspensory  apparatus  of  the 
parabasal  body  is  here  relatively  larger  and  apparently  more  highly 
developed  than  in  any  of  the  other  flagellates.  A  few  of  the  trypani- 
form stages  also  show  a  similar  parabasal  rhizoplast,  according  to  the 
figures  of  Chagas  (1909).  In  the  crithidial  stages  of  this  trypano- 
some,  as  shown  in  figure  A,  5,  there  is  a  sharply  defined  nuclear  rhizo- 
plast passing  from  the  centriole  of  the  nucleus  to  the  blepharoplast. 
The  position  of  the  parabasal  body  in  8.  cruzi  is  similar  to  that  of 
Crithidia  euryophthalmi  and  is  situated  apparently  to  one  side  of  the 
nuclear  rhizoplast. 

One  of  the  most  important  discoveries  in  connection  with  the  study 
of  these  organelles  is  the  fact  that  the  parabasal  body,  or  the  so-called 
kinetonucleus,  is  not  axial  in  position  in  Crithidia  euryophthalmi 
(figs.  B,  1-4)  and  apparently  not  in  the  other  flagellates  (figs.  A, 
1,  3-5).  With  the  evidence  at  hand  to  show  definitely  that  this  body 
is  not  axial  but  a  lateral  appendage  of  the  blepharoplast  in  C.  euryo- 
phthalmi it  is  necessary  to  look  upon  this  organelle  as  something  other 
than  a  second  nucleus  or  a  kinetonucleus.  Reference  has  already 
been  made  to  the  work  of  Dr:  Kofoid  (1916)  concerning  the  homology 
of  this  organelle  with  the  parabasal  body  of  other  flagellates.  The 
observations  concerning  the  origin  of  this  structure,  the  location 
and  relation  of  the  parabasal  body  to  the  other  organelles  as  found 
in  the  study  of  C.  euryophthalmi  offer  some  of  the  best  evidence  against 
the  binuclear  conception  of  these  flagellates  (Hartmann,  1911). 

Flagellum  and  undulating  membrane. — Owing  to  the  relationship 
which  exists  between  the  flagellum  (/?.)  and  undulating  membrane 
(und.  m.)  it  is  convenient  to  describe  these  two  organelles  together. 
The  flagellum  consists  of  an  outgrowth  from  the  blepharoplast  and  is 
surrounded  by  a  cytoplasmic  sheath  which  is  continuous  with  the  sack- 
like  sheath  covering  the  parabasal  body.  In  the  ordinary  preparations 
no  distinction  can  be  made  between  this  central  portion  originating 


152  University  of  California  Publications  in  Zoology       [VOL.  19 

from  the  blepharoplast  and  the  cytoplasmic  sheath  surrounding  it. 
The  entire  flagellum  stains  as  a  single  heavy  line,  almost  chromatoidal 
in  appearance.  It  does  not  stain  so  deeply  as  does  the  nucleus,  but 
much  deeper  than  the  cytoplasm  of  the  body  and  of  the  undulating 
membrane.  As  the  flagellum  forms  and  lengthens,  there  is  an  accom- 
panying elongation  of  the  protoplasm  which  forms  the  undulating 
membrane  (und.  m.).  Both  endoplasm  and  ectoplasm  enter  into  the 
formation  of  this  organelle.  Usually  there  is  a  thin,  narrow  band  of 
clear  ectoplasm  lying  parallel  to  the  flagellum  (fig.  A,  5).  The  length 
of  the  membrane,  and  consequently  of  the  intracellular  part  of  the 
flagellum,  is  greater  among  the  crithidias  of  the  stomach  phase  than  of 
the  rectal  phase  owing  to  the  shifting  of  the  cytoplasm  of  the  body 
in  the  transition.  This  is  usually  true  also  of  the  crithidias  as  com^ 
pared  with  the  crithidial  stages  of  the  trypanosomes  (fig.  A,  1,  2,  3,  4). 
As  previously  noted,  the  length  of  the  free  flagellum  (fig.  A,  3)  has 
no  significance  from  the  viewpoint  of  comparative  morphology,  since 
there  is  a  wide  variation  in  the  length  of  this  organelle  for  each  species 
of  Crithidia  and  of  Trypanosoma. 


THE  LIFE  CYCLE  OF  CRITHIDIA  EURYOPHTHALMI 

The  life  cycle  of  C.  euryophthalmi  in  Euryophthalmus  convivus 
begins,  so  far  as  known,  with  the  casual  ingestion  with  food  of 
spores  from  the  fecal  matter  of  infected  insects.  E.  convivus  com- 
monly feeds  upon  the  sap  from  the  growing  tips  of  the  lupine,  which 
show  many  indications  of  excreta.  In  these  same  regions  of  the  lupine 
galls  and  other  abnormal  growths  occur  in  great  abundance.  The  pos- 
sibility that  these  insects  were  getting  their  infection  of  C.  euryo- 
phthalmi  from  the  sap  of  the  lupine  was  suggested  by  the  work  of 
Franga  (1914).  Examinations  of  the  sap  of  the  lupine  were  accord- 
ingly made.  Nematodes,  numerous  yeast-like  spores,  and  bacteria 
were  found.  No  organisms  were  discovered,  however,  of  any  descrip- 
tion which  could  be  linked  to  the  known  stages  of  C.  euryophthalmi 
in  the  digestive  tract  of  the  host. 

The  large  number  of  parasites  in  the  life  cycle  of  C.  euryophthalmi 
can  be  grouped  readily  into  two  series :  the  developmental,  or  infective, 
and  the  degenerative,  which  are  comparable  to  the  developmental  and 
degenerative  series  of  T.  lewisi  in  the  flea,  as  described  by  Minchin 
and  Thomson.  That  the  correlation  between  these  two  life  cycles  is 
marked  will  become  clear  in  following  the  discussion  of  the  life  cycle 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma        153 

of  C.  euryophthalmi,  notwithstanding  the  fact  that  the  initial  stages 
of  the  two  life  cycles  are  entirely  different  in  the  insects.  The  initial 
stages  of  the  crithidial  life  cycle  are  small,  oval  spores,  which  develop 
into  elongate  Crithidial  flagellates,  whereas  the  initial  stages  of  the 
trypanosome  are  trypanosomes  from  the  blood  of  a  rat,  which  indirectly 
by  a  process  of  multiple  fission,  produce  very  similar  elongate  crithidial 
flagellates.  Subsequently  all  of  the  rectal  stages  in  each  life  cycle  are 
very  similar. 


Fig.  B.  Five  figures  of  Crithidia  euryophthalmi  to  show  the  relation  of  the 
parabasal  body  to  the  flagellum,  blepharoplast,  rhizoplast,  and  nucleus.  X  3500. 
Abbreviations  same  as  in  figure  A. 

The  developmental  or  infective  series  of  Crithidia  euryophthalmi 
consists  of  the  stomach  phase  or  the  crithidias  of  the  ''crop,"  mid- 
stomach  and  upper  part  of  the  pyloric  expansion.  The  degenerative 
series  includes  the  late  rectal  phase  or  the  attached  crithidias  of  the 
pyloric  expansion. 


THE  DEVELOPMENTAL  SERIES 
STOMACH  PHASE 

The  stomach  phase  of  Crithidia  euryophthalmi  is  apparently  init- 
iated when  the  small,  oval  spores  (pi.  2,  figs.  1,  2)  casually  ingested- 
with  food  begin  to  develop  in  the  "crop."     Such  initial    infective 
spores  develop  rapidly  into  a  swarm  of  multiplying  crithidias  (pi.  2, 
figs.  2-7).    Later  as  mature  flagellates  (pi.  2,  figs.  9,  10)  the  crithidias 


154  University  of  California  Publications  in  Zoology        [VOL.  19 

may  be  carried  immediately  to  the  pyloric  expansion  through  the  mid- 
stomach  by  the  current  of  food,  or  they  may  undergo  a  process  of 
multiple  fission  extracellularly  or  intracellularly  in  the  "crop."  The 
process  of  multiple  fission  may  be  similar  to  that  of  the  spheres  of 
Trypanosoma  leivisi  (Minchin  and  Thomson,  1915),  which  have  certain 
characteristics  in  common  with  the  somatella  in  the  multiple  fission  of 
some  of  the  Polymastigina  (trichomonad  flagellates)  described  by 
Kofoid  and  Swezy  (1915).  In  addition  to  this  type  of  multiple  fission 
there  is  a  second  and  entirely  different  process  of  multiple  fission,  the 
internal  or  endogenous  budding  (pi.  2,  figs.  11-23).  These  two  pro- 
cesses of  multiple  fission  have  been  described  briefly  in  a  preliminary 
communication  (McCulloch,  1917).  Endogenous  budding  has  been 
emphasized  in  this  paper  and  described  in  detail  because  of  its  interest 
and  importance. 


EXTRACELULAR   CRITHIDIAS 

Oval  spores. — The  initial  infective  spores  (pi.  2,  fig.  1)  are  ovoid 
and  stain  deeply.  They  are  found  in  small  numbers  in  the  "crop," 
and  present  several  distinguishing  marks  which  serve  as  a  means  of 
identification.  They  average  1.7/>t  in  width  and  about  3.4/A  in  length. 
The  nucleus  stains  diffusely  and  forms  a  solid  mass  of  chromatin  at 
the  extreme  posterior  end  of  the  body.  The  parabasal  body  lies  within 
the  anterior  half  of  the  spore,  about  equally  distant  from  the  nucleus 
and  forward  end.  One  end  of  this  bar-shaped  structure,  or  parabasal 
body,  lies  close  to  the  thick  periplast.  On  all  the  other  sides  of  this 
organelle  there  is  the  characteristic  light  area,  which  quickly  destains 
in  iron-haematoxylin  preparations.  Careful  focusing  has  revealed  a 
faint  nuclear  rhizoplast  passing  from  the  nucleus  toward  the  region 
of  the  parabasal  body. 

Developing  crithidias. — When  the  initial  infective  spores  begin 
to  develop  in  the  "crop"  a  change  occurs  in  their  shape  and  staining 
capacity  (pi.  2,  fig.  9).  The  forward  outgrowth  of  the  flagellum 
assists  in  the  elongation  of  the  anterior  end  and  the  formation  of  the 
undulating  membrane  (pi.  2,  figs.  6-9).  The  posterior  region  elon- 
gates less  rapidly,  but  in  time  it  frequently  attains  an  even  greater 
length  than  the  anterior  end  (pi.  2,  fig.  9).  The  length  of  the  free 
flagellated  crithidias  which  result  from  the  developing  forms  varies 
greatly  at  all  times  regardless  of  their  location  in  the  digestive  tract. 
In  figure  9,  plate  2,  the  crithidial  flagellate  has  reached  the  extreme 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma        155 

length,  comparatively,  of  32/z.  At  the  other  extreme,  a  short  crithidia 
of  7ju  (pi.  2,  fig.  12)  is  evidently  mature,  since  it  is  undergoing  a 
process  of  multiple  fission  of  the  endogenous-budding  type.  Between 
these  extremes  is  a  series  of  intergrading  forms.  The  sizes  and 
shapes  of  a  swarm  of  free  crithidial  flagellates  from  the  life  cycle 
of  anyone  of  these  species  of  flagellates  under  discussion  show  great 
diversity,  and  Crithidia  euryophthalmi  is  not  an  exception,  as  indi- 
cated in  plates  2  and  4.  The  change  in  the  appearance  of  the  nucleus 
as  development  proceeds  is  noticeable.  The  deeply  staining  mass  of 
chromatin  (pi.  2,  figs.  1,  2)  becomes  a  nucleus  containing  a  prominent 
central  karyosome  and  a  chromatin-encrusted  nuclear  membrane  (pi. 
2,  figs.  3-10).  Between  the  karyosome  and  membrane  is  a  clear  area, 
which  destains  very  readily  after  iron-haematoxylin. 


MULTIPLE  FISSION 

Endogenous  budding. — In  looking  over  a  large  number  of  prepara- 
tions of  the  digestive  tract  of  Euryopkthalmus  convivus,  in  the  early 
part  of  1916,  I  chanced  upon  a  splendid  preparation  of  a  "crop" 
from  a  nymph  which  contained  an  exceedingly  heavy  infection  of 
crithidias  of  all  shapes  and  sizes.  The  smear  was  well  fixed  and  well 
stained. 

Among  other  things  the  nuclear  structure  was  studied  in  detail 
to  determine  whether  the  nucleus  of  these  crithidias  divided  by  a 
mitotic  process  or  by  a  more  primitive  method  of  mitosis.  This  search 
led  to  the  discovery  of  a  flagellate  which  apparently  contained  two 
nuclei  (pi.  2,  fig.  12),  in  a  linear  arrangement  with  respect  to  the  long 
axis  of  the  body.  Shortly  after  another  crithidia  (pi.  2,  fig.  13)  was 
found  in  the  same  preparation,  containing  apparently  three  similar 
nuclei,  which  were  arranged  in  a  like  linear  series.  In  the  latter 
(pi.  2,  fig.  13)  careful  focusing  revealed  the  outline  of  a  fourth 
partially  concealed  beneath  the  most  anterior  nuclear  structure.  In 
each  of  these  flagellates  (pi.  2,  figs.  12,  13)  no  indications  of  any 
ordinary  process  of  binary  fission  were  detected.  The  blepharoplast, 
parabasal  body,  and  the  rhizoplasts  in  each  were  still  intact  in  so  far 
as  could  be  determined.  The  alternative  hypothesis  that  these  nucleus- 
like  structures,  which  are  relatively  small,  are  internal  parasites  of  a 
bacterial  or  protozoan  nature  naturally  was  given  full  consideration. 
Drawings  of  these  flagellates  were  made  with  the  camera  lucida  and 
the  readings  were  taken  for  future  reference.  Owing  to  the  abun- 


156  University  of  California  Publications  in  Zoology        [VOL.  19 

dance  of  the  parasites  in  this  preparation  of  the  "crop"  ample 
material  was  at  hand  for  an  extensive  study  of  the  morphology  of  this 
phase  of  the  life  cycle  of  Crithidia  euryophthalmi.  From  time  to 
time  more  flagellates  were  observed,  apparently  multinucleated,  but 
little  additional  light  was  thrown  upon  their  peculiar  nuclear  struc- 
tures until  a  large  flagellate,  such  as  shown  in  figure  22,  plate  1,  was 
observed.  This  crithidia  was  relatively  large  and  contained  approxi- 
mately twelve  '  *  binucleated "  spore-like  forms.  The  structure  of  the 
sporelike  forms  within  the  periplast  of  the  large  flagellate  was  similar 
to  that  of  the  numerous  small,  oval  spores  in  the  immediately  sur- 
rounding field,  which  were,  beyond  doubt,  stages  of  the  life  cycle  of 
C.  euryophthalmi.  Another  large  flagellate  containing  six  nuclear 
structures  within  the  periplast  (pi.  2,  fig.  21)  was  also  drawn.  The 
flagellum  and  parabasal  body  were  clearly  outlined,  as  in  the  former, 
multinucleated  flagellates.  Some  of  the  enclosed  nuclear-like  struc- 
tures were  deeply  stained,  owing  to  the  thickness  of  the  preparation, 
but  the  majority  presented  the  same  appearance  as  did  the  nuclear- 
like  structures  in  figures  12  and  13. 

Investigation  of  the  smear  revealed  more  and  more  evidence  of  a 
possible  endogenous,  or  internal  budding,  in  the  life  cycle  of  Crithidia 
euryophthalmi.  Past  experience  indicated  that  the  preparations  of 
the  ' '  crops ' '  of  young  nymphs  furnished  the  best  smears  for  the  study 
of  the  initial  infections,  of  which  the  endogenous  budding  forms 
were  evidently  a  part.  An  effort  was  accordingly  made  during  the 
next  breeding  season  of  Euryophthalmus  convivus  to  collect  as  many 
young  nymphs  as  possible  in  order  to  obtain  additional  prepara- 
tions of  the  digestive  tract,  with  reference  to  the  * t  crop ' '  in  particular. 

Out  of  a  large  number  of  preparations  of  the  "crops"  prepared 
during  the  following  season,  only  two  contained  additional  stages  of 
the  process  of  endogenous  budding.  The  percentage  of  infection  of 
the  "crops"  of  young  nymphs  was  found  to  be  approximately  twenty 
per  cent  as  compared  with  two  per  cent  among  adult  insects. 

In  the  two  preparations  there  were  numerous  small,  "binucleated," 
spore-like  forms,  grouped  near  discarded  flagella,  with  or  without  ble- 
pharoplasts,  and  with  parabasal  bodies  still  attached  (pi.  2,  fig.  23). 
It  was  easy  to  conceive  of  the  degeneration  of  the  cytoplasm  surround- 
ing the  spore-like  forms,  leaving  a  field  covered  with  the  internal 
spores,  or  zooids,  and  the  extranuclear  organelles  of  the  parent  cell. 
In  the  earlier  stages  of  degeneration  presumably  the  parabasal  bodies 
were  still  attached  by  the  parabasal  rhizoplasts  to  the  blepharoplasts 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma        157 

at  the  bases  of  the  flagella.  Later  stages  showed  only  flagella  among 
the  zooids.  This  additional  evidence  from  the  two  smears  at  once 
suggested  the  work  of  Moore  and  Breinl  (1907),  in  which  "  latent 
bodies ' '  were  described  in  the  life  cycle  of  a  haemoflagellate,  Trypano- 
soma gambiense. 

According  to  these  investigators  T.  gambiense  in  the  blood  of  a 
vertebrate  host  formed  latent  bodies  in  the  nuclear  region,  under 
certain  conditions.  These  latent  bodies  are  pictured  and  described 
as  being  small  nucleated  structures  having  a  nuclear  membrane  and  a 
single  mass  of  chromatin  in  the  center.  The  exact  method  of  the 
formation  of  the  bodies  in  the  nucleus  or  from  the  nucleus  is  not 
clear,  since  none  of  the  early  stages  in  the  formation  of  the  latent 
bodies  are  figured  or  described  in  detail.  Apparently  only  the  results 
of  an  endogenous  process  in  the  life  cycle  of  T.  gambiense  were  ob- 
served by  these  investigators.  The  final  stages  of  the  process  in  the 
life  cycle  of  T.  gambiense  are  the  presence  of  large,  degenerating  try- 
panosomes  with  minute,  nucleated  latent  bodies  in  the  nuclear  area. 
The  flagellum,  kinetonucleus,  or  parabasal  body  and  cytoplasm  are  in 
various  stages  of  degeneration.  Their  figures  of  the  latent  bodies 
(Moore  and  Breinl,  1907)  should  be  compared  with  figure  23  of  this 
paper. 

The  latent  bodies  in  the  life  cycle  of  Trypanosoma  gambiense  and 
the  endogenous  zooids  of  the  life  cycle  of  Crithidia  euryophthalmi  are 
not  the  only  instances  of  an  endogenous  budding  in  the  life  cycle  of 
the  Protomonadina  (Trypanosoma,  Crithidia,  Herpetomonas,  Lepto- 
monas).  Minchin  and  Thomson  (1915)  searched  for  evidence  of  an 
endogenous  budding  in  the  life  cycle  of  T.  lewisi  but  failed  to  find  any 
indications  of  the  process.  However,  in  the  life  cycle  of  Leptomonas 
pattoni,  one  of  the  so-called  natural  flagellates  frequently  found  in 
the  digestive  tract  of  the  flea,  they  found  several  crithidial-like  flagel- 
lates containing  a  nucleus  and  an  endogenous  bud.  These  authors 
suggested  that  the  endogenous  buds  found  therein  were  doubtless  com- 
parable to  the  latent  bodies  of  Moore  and  Breinl. 

The  early  stages  of  the  endogenous,  or  internal  budding  of  Crith- 
idia euryophthalmi  are  shown  in  figures  11  and  12,  plate  2.  In  figure 
11  there  is  a  relatively  short  flagellate  undergoing  multiple  fission  in 
this  way.  The  nucleus  has  budded  off  two  circular,  nucleus-like  buds, 
each  showing  a  chromatin-encrusted  nuclear  membrane.  The  para- 
basal  body  and  the  flagellum  of  this  flagellate  are  still  intact  and  have 
no  indications  of  binary  fission.  Somewhat  similar  to  this  flagellate 


158  University  of  California  Publications  in  Zoology       [VOL.  19 

is  the  one  shown  in  figure  12,  plate  2.  The  anterior  end  of  the  body 
is  slightly  shortened  but  the  nuclear  structure  has  the  same  general 
appearance.  Only  one  bud  has  been  given  off,  and  this  lies  directly 
posterior  to  the  nucleus  proper.  As  in  the  former  case,  the  chromatin 
is  collected  on  the  inner  surface  of  the  nuclear  membrane  of  both  the 
nucleus  and  endogenous  bud.  In  figure  13,  plate  2,  a  more  complex 
organization  was  observed.  The  nucleus  and  two  clearly  defined 
nuclear  buds  are  arranged  in  a  linear  series,  the  two  buds  being  pos- 
terior to  the  nucleus  proper.  Partially  concealed  by  the  nucleus  is  a 
third  bud,  which  is  being  constricted  off  from  the  nucleus.  This  par- 
ticular view  of  the  process  is  in  all  probability  an  end  view  and  only 
a  portion  of  the  bud  is  visible.  If  the  observations  and  interpretation 
of  this  structure  be  correct,  the  nucleus,  with  all  of  its  chromatin  col- 
lected on  the  inner  surface  of  the  membrane,  repeatedly  constricts  or 
buds  off  a  portion,  forming  a  series  of  nuclear  buds  (pi.  2,  figs.  13, 
20,  21).  There  is  at  hand  at  present  no  evidence  of  a  central  karyo- 
some  being  present  in  the  nucleus  when  endogenous  buds  are  formed. 
The  nuclear  buds,  therefore,  in  so  far  as  our  observations  have  gone, 
are  due  neither  to  a  clearly  defined  amitosis  nor  to  a  primitive  form 
of  mitosis  of  the  chromatin  material  which  normally  occurs  in  a 
central  karyosome. 

Although  evidence  of  a  mitotic  process  in  this  nuclear  division  is 
wanting,  nevertheless  some  evidence  of  a  promitosis  has  been  found  in 
the  division  of  the  nucleus  in  an  early  stage  of  the  formation  of  a  soma- 
tella  (pi.  3,  fig.  25).  The  formation  of  the  somatella  involving  the 
" rounding  up "  of  an  elongate  flagellate  into  a  sphere  will  be  described 
shortly;  it  will  suffice  for  our  purpose  here  to  point  out  salient  fea- 
tures of  the  internal  structure  of  the  ''rounding  up"  flagellate  (pi.  3, 
fig.  25).  The  blepharoplast,  parabasal  body,  and  the  rhizoplast  of  this 
flagellate  have  not  yet  begun  to  divide  but  the  nucleus  is  beginning  to 
form  two  daughter-nuclei.  The  central  karyosome  which  is  normally 
present  in  C.  euryophthalmi  has  formed  two  unequal  masses  of  chro- 
matin connected  by  a  centrodesmose.  No  critical  evidence  was  found 
showing  that  there  is  present  at  each  end  of  the  controdesmose  a  cent- 
riole  or  centrosome  differentiated  from  the  chromatin  material  in  this 
minute  form.  Peripherally  there  is  the  nuclear  membrane  still  present 
and  intact,  but  it  is  constricting  in  the  center  to  form  two  nuclei.  If  a 
similar  division  of  the  chromatin  material  occurs  in  the  nuclear  bud- 
ding it  has  thus  far  escaped  observation.  The  number  of  early  stages 
of  nuclear  budding  studied  has  been  small,  owing  to  the  rarity  of 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma        159 

preparations  showing  the  process.  In  every  instance  of  nuclear  bud- 
ding so  far  observed  the  chromatin  has  been  peripheral,  with  a  more 
or  less  unequal  distribution  on  the  nuclear  membrane.  Frequently 
there  is  one  heavy  mass  with  a  uniform  amount  elsewhere  on  the 
membrane  (pi.  2,  fig.  15). 

A  somewhat  larger,  elongate  flagellate,  with  sharply  defined  endo- 
genous buds,  is  shown  in  figure  14,  plate  2.  Unlike  the  endogenous 
forms  just  described  the  buds  here  are  not  posterior  to  the  nucleus. 
In  figure  14,  opposite  the  parabasal  body,  there  is  a  bud  decidedly 
anterior  and  lateral  in  position.  A  second  endogenous  bud  is  also  ante- 
rior between  the  nucleus  and  parabasal  body.  Within  all  of  these 
nuclear  structures  the  chromatin  is  massed  irregularly  upon  the  nuclear 
membrane. 

In  addition  to  these  elongate  flagellates  undergoing  multiple  fission 
of  this  type  there  are  also  pear-shaped  crithidias  (pi.  2,  figs.  15-18) 
containing  several  or  numerous  endogenous  buds.  Possibly  it  is  the 
greater  thickness  of  these  flagellates,  with  consequent  decrease  in 
destaining  capacity,  which  makes  the  buds  within  stand  out  so  dis- 
tinctly. Another  marked  characteristic  of  the  pear-shaped  flagellates 
is  the  lack  of  differentiation  between  the  nucleus  and  the  buds.  There 
is  no  evidence  of  a  parent  nucleus  which  has  given  rise  to  the  buds. 
The  distribution  of  the  chromatin  within  these  buds  presents  some 
interesting  variations.  In  figure  15  the  buds  contain  a  clearly  defined 
chromatin  encrusted  nuclear  membrane  together  with  one  distinct  mass 
or  chromatin  granule.  In  figure  17  the  massing  of  the  chromatin  may 
mean  a  more  advanced  stage  since  it  is  no  longer  on  the  entire  nuclear 
membrane  but  has  been  segregated  into  two  masses,  which  give  the 
buds  a  bipartite  appearance.  Proceeding  to  figure  18  in  this  pear- 
shaped  individual  more  steps  in  advance  are  portrayed,  namely  the 
unused  portions  of  the  flagellate  are  beginning  to  degenerate  around 
the  endogenous  buds.  The  parabasal  body  has  already  disappeared, 
and  only  a  portion  of  the  discarded  flagellum  remains  near  by.  The 
structure  of  the  endogenous  buds  of  this  spherical  crithidia  is  remark- 
ably uniform.  In  each  bud  the  chromatin  granule  is  adherent  to 
the  nuclear  membrane. 

Other  elongate  flagellates  undergoing  multiple  fission  are  observed 
in  figures  19,  20,  and  21.  Some  interesting  variations  can  be  pointed 
out  in  these  crithidias.  In  figure  19  there  is  a  nuclear  rhizoplast, 
which  can  be  traced  from  the  edge  of  the  nuclear  membrane  to  the 
blepharoplast.  Of  greater  interest  are  the  variations  shown  in  figures 


160  University  of  California  Publications  in  Zoology       [VOL.  19 

20  and  21.  In  figure  20  the  binucleated  appearance  of  the  nucleus  and 
buds  is  very  prominent.  The  chromatin  granules  have  a  paired  effect 
which  is  difficult  to  interpret.  In  figure  21  a  more  complex  structure 
is  represented.  Anterior  to  the  nucleus  there  is  a  group  of  five 
endogenous  buds.  Two  of  these,  owing  to  their  position,  are  not  de- 
stained  sufficiently  to  make  clear  their  nuclear  structure.  This  large 
flagellate  has  a  sharply  defined  parabasal  body  and  flagellum,  similar 
to  those  in  figures  19  and  20.  The  large  size  of  such  forms  has  aided 
materially  in  the  interpretation  of  the  endogenous  buds,  which,  upon 
developing,  form  the  so-called  zooids. 

The  most  mature  stage  of  endogenous  budding  yet  found,  wherein 
the  resulting  zooids  are  still  within  the  periplast,  is  pictured  in 
figure  22.  There  are  approximately  twelve  clearly  defined  zooids 
massed  near  the  central  part  of  this  large  flagellate.  The  structure 
of  the  zooids  could  be  studied  readily.  The  nucleus  and  parabasal 
body  in  each,  because  of  their  deep  stain,  helped  to  distinguish  the 
several  zooids.  The  other  organelles  were  not  visible.  Another  inter- 
esting feature  concerning  these  zooids  is  the  difference  in  the  stages 
of  their  development.  Some  are  larger  and  more  mature  and  were 
doubtless  budden  off  from  the  nucleus  first.  Probably  the  zooids  of 
the  flagellate  have  been  retained  within  the  periplast  of  the  parent 
for  a  longer  period  than  usual.  If  the  size  of  these  zooids  be  compared 
with  that  of  the  free  zooids  shown  in  figure  23  the  former,  on  the 
whole,  are  larger  and  more  fully  developed.  There  are,  moreover, 
no  marked  signs  of  degeneration  of  the  parabasal  body,  of  the  flagel- 
lum, or  of  the  cytoplasm.  The  parabasal  body  is  almost  hidden  by 
the  zooids  in  that  region. 

The  final  step  in  the  endogenous-budding  process  comes  with  the 
degeneration  of  the  body-plasm  of  the  parent  flagellate,  leaving  a  mass 
of  zooids,  together  with  the  flagellum  and  parabasal  body  of  the  parent. 
Portions  of  smears  have  been  observed  to  be  literally  covered  with 
zooids  and  discarded  flagella.  As  degeneration  proceeds  the  parabasal 
bodies  next  disappear,  and  finally  the  flagella,  leaving  only  numerous 
zooids  of  various  sizes  and  structure.  The  last  phase  is  the  one  most 
frequently  observed  in  preparations  of  Crithidia  euryophthalmi.  I 
had  been  working  with  C.  euryophthalmi  more  or  less  for  a  period 
of  almost  two  years  before  the  clue  as  to  the  origin  of  these  small 
zooids  was  found.  It  had  been  most  puzzling  to  find  so  many  of  these 
small,  non-flagellated,  binucleated  forms  (pi.  4,  fig.  40),  which  were 
obviously  unlike  the  initial  infective  spores  (pi.  2,  figs.  1,  2).  They 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma        161 

were  smaller  in  size,  their  periplast  was  thinner,  and  they  destained 
much  more  rapidly  after  iron-haemotoxylin.  Serial  sections  of  the 
mid-stomach  showed  them  grouped  in  pockets  between  the  epithelial 
cells.  Possibly  the  endogenous  process  occurred  there  or  they  were 
lodged  there  as  the  current  of  food  carried  the  others  down  into  the 
pyloric  expansion.  They  could  be  arranged  in  a  series,  beginning 
with  those  averaging  about  1.7/x,  in  length  (pi.  4,  fig.  40)  and  gradating 
to  the  size  of  the  spores  which  were  regarded  as  the  initial  infective 
spores  (pi.  2,  fig.  1;  pi.  4,  figs.  40-54). 

The  development  of  the  endogenous  buds  into  binucleated  zooids 
is  not  easy  to  interpret.  In  figure  23,  plate  2,  the  numerous  zooids 
present  variations  in  both  size  and  structure.  Unlike  the  latent  bodies 
of  Trypanosoma  gambiense  of  Moore  and  Breinl  (1907)  and  the  endo- 
genous buds  of  Leptomanas  pattoni  of  Minchin  and  Thomson  (1915), 
these  endogenous  buds  show  no  central  chromatin  granule  within  the 
nuclear  membrane.  Their  chromatin  is  distributed  at  the  periphery 
of  the  nucleus  usually  in  one  of  three  ways.  In  the  elongate  flagellates 
(pi.  2,  figs.  11,  14,  19)  the  buds  have  their  chromatin  material  massed 
irregularly  on  the  nuclear  membrane.  A  second  form  of  peripheral 
chromatin  in  the  nucleus  is  a  noticeable  mass  or  granule  at  one  point. 
This  is  characteristic  of  the  buds  within  the  pear-shaped  flagellates 
(pi.  2,  figs.  15-18).  The  third  type  of  nuclear  structure  in  the  buds 
is  possibly  only  a  slight  modification  of  the  others.  In  figure  20, 
plate  2,  the  internal  or  endogenous  buds  appear  to  be  binucleated 
because  of  the  peculiar  segregation  of  the  chromatin  material  on  the 
nuclear  membrane.  At  present  these  several  modifications  of  the 
nuclear  structure  are  not  regarded  as  having  any  special  sequence  or 
significance.  In  some  of  the  smallest  zooids  of  figure  23  there  are 
still  other  modifications  wherein  a  single,  deeply  staining  mass  is  fre- 
quently observed.  Owing  to  the  size  of  these  zooids  it  is  extremely 
difficult,  even  with  a  binocular  microscope,  to  form  any  adequate 
conception  of  their  structure. 

A  diligent  search  has  been  made  among  these  small  forms  to  find 
a  series  of  developing  zooids  which  would  clearly  show  the  whole 
process  of  the  formation  of  the  several  organelles.  While  Crithidia 
euryophthalmi  undoubtedly  furnishes  the  necessary  material  for  such 
a  study,  the  size  of  the  endogenous  buds  makes  the  interpretation 
exceedingly  difficult.  It  is  therefore  disappointing  that  a  complete 
series  has  not  yet  been  accumulated  showing  the  steps  which  are 
thought  to  take  place  in  the  development  of  a  bud  into  a  typical 


162  University  of  California  Publications  in  Zoology       [VOL.  19 

crithidia.  The  results  of  the  study  now  indicate  that  the  extranuclear 
organelles  are  apparently  formed  as  outgrowths  from  the  nucleus. 
It  is  conceived  that  the  centrosome  of  the  nucleus  divides,  giving  rise 
to  the  extranuclear  centrosome,  or  blepharoplast.  From  the  blepharo- 
plast,  which  is  the  dynamic  center  of  the  nuclear  outgrowth,  two 
other  organelles  are  formed.  The  flagellum  grows  forward  anteriorly 
and  the  parabasal  body  to  the  side.  The  connection  which  persists 
between  the  blepharoplast  and  the  nucleus  is  the  nuclear  rhizoplast. 
Between  the  blepharoplast  and  the  parabasal  body  is  the  parabasal 
rhizoplast.  Careful  observation  of  some  of  the  larger  forms  reveals 
a  cytoplasmic  sheath  around  the  flagellum,  which  is  continuous  with 
a  similar  sheath  around  the  parabasal  body.  The  latter  sheath  is 
like  a  sack,  in  which  the  deeply  staining,  bar-shaped  parabasal  body 
is  suspended.  The  variation  in  size  of  the  parabasal  body  may 
explain  the  light  area  which  is  frequently  observed  to  surround  this 
organelle.  The  fan-shaped  appearance  of  the  parabasal  rhizoplast 
is  due  in  all  probability  to  this  sacklike  sheath.  Any  general  con- 
clusions concerning  the  origin  of  these  organelles  would,  at  this  time 
however,  be  premature,  although  the  observations  of  the  zooids  under 
the  binocular  microscope  tend  to  give  this  conception  of  their  origin. 
It  is  necessary  to  keep  in  mind  constantly  the  fact  that  the  material 
with  which  we  are  dealing  is  the  complex  life  cycle  of  a  flagellate  and 
that  several  stages  of  the  life  cycle  have  not  yet  been  followed,  step 
by  step,  in  the  living  material.  The  possibility  of  confusing  two  life 
cycles  is  always  present,  and  the  fact  that  all  of  the  work  is  done 
near  the  limits  of  microscopical  magnification  adds  further  possibility 
of  misconception. 

Aside  from  these  difficulties  and  doubtful  points,  however,  the 
discovery  of  all  of  these  stages  of  what  has  been  interpretated  as  a 
process  of  endogenous  budding,  opens  up  further  problems  for  investi- 
gation in  the  life  cycles  of  these  flagellates.  The  origin  of  the  para- 
basal body  in  the  endogenous  bud  is  a  big  problem  in  itself.  In  addi- 
tion, the  light  thrown  upon  the  probable  origin  of  the  numerous 
binucleated  spores  or  Leishmanw-like  bodies,  which  occur  so  abun- 
dantly in  the  life  cycles  of  such  flagellates  as  Schizotrypanum  cruzi 
(Chagas,  1910),  Crithidia  melophagia  (Porter,  1910),  and  C.  lepto- 
coridis  (McCulloch,  1915),  is  very  suggestive. 

Somatella. — Previous  to  the  discovery  of  the  multinucleated  flag- 
ellates which  were  undergoing  a  process  of  internal  or  endogenous 
budding,  another  type  of  multiple  fission  had  been  studied  in  prepara- 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma        163 

tions  of  the  "crop"  of  Euryopthalmus  convivus,  namely,  that  which 
leads  to  the  formation  of  the  somatella  (pi.  3,  figs.  28-32).  These 
spherical  crithidias  have  certain  characteristics  in  common  with  the 
tailed  and  tailless  spheres  described  in  the  life  cycle  of  Trypanosoma 
lewisi  by  Minchin  and  Thomson  (1915).  In  a  general  way  they  re- 
semble also  the  somatellas  in  the  life  cycle  of  some  of  the  Polymastigina 
described  by  Kofoid  and  Swezy  (1915).  Beginning  with  the  earliest 
stages  of  this  type  of  multiple  fission  a  series  of  flagellates  can  be  ar- 
ranged which  parallels  the  successive  stages  of  multiple  fission  of  T. 
lewisi  (cf.  Minchin  and  Thomson,  1915,  pi.  37).  At  the  beginning 
of  this  series  such  flagellates  as  shown  in  figures  24  and  25  of  plate  3 
are  to  be  found.  In  figure  24  the  elongate  flagellate  is  beginning  to 
round  up.  The  attenuate  ends  are  being  drawn  up  to  the  central  part 
of  the  body  and  the  flagellum  has  become  intracellular  throughout  its 
length.  A  somewhat  different  formation  of  the  sphere  is  shown  in 
figure  25,  plate  3.  The  long,  attenuate  ends  are  being  wrapped  about 
the  body  and  the  flagellum  is  likewise  entirely  intracellular  in  this 
flagellate.  Other  important  features  to  be  observed  in  connection 
with  this  flagellate  are  the  first  indications  of  nuclear  division  in  the 
spherical  formation.  The  centriole  or  centrosome  within  the  nucleus 
of  the  rounding-up  flagellate  is  initiating  the  division  of  the  organ- 
elles.  It  has  divided  into  two  daughter-centrosomes,  which  are  still 
connected  by  a  centrodesmose,  and  with  each  daughter-centrosome  there 
is  present  a  varying  amount  of  chromatin  material.  No  chromosomes 
are  present  at  this  stage  of  the  division,  and  their  presence  at  any 
period  throughout  the  process  in  Crithidia  euryophthalmi  has  not  been 
established.  Indications  of  chromosomes  in  the  ordinary  binary  fission 
have  been  observed,  but  no  definite  number  has  been  determined.  The 
nuclear  membrane  has  begun  to  constrict  on  either  side  of  the  centro- 
desmose. The  entire  division  of  the  chromatin  material  takes  place 
within  the  nuclear  membrane,  and  the  process  is  apparently  a  primi- 
tive type  of  promitosis.  The  centrosome  within  the  karyosome  is  not 
always  the  first  to  divide ;  on  the  contrary,  in  many  instances  the  first 
indication  of  the  division  of  the  organelles  is  shown  by  the  blepharo- 
plast  or  extranuclear  centrosome.  When  the  blepharoplast  divides 
the  division  of  the  parabasal  body  follows  immediately.  A  repetition 
of  the  division  of  the  several  organelles  occurs  and  the  spheres  finally 
break  up  into  a  number  of  merozoites,  or  daughter  individuals. 

The  spherical  formation  is  completed  in  figure  26,  plate  3.     A 
flagellum  is  protruding  beyond  the  surface  of  the  sphere.    Internally 


164  University  of  California  Publications  in  Zoology       [VOL.  19 

division  of  the  blepharoplast,  the  parabasal  body,  and  the  nucleus 
has  occurred.  A  second  flagellum  growing  out  from  the  daughter- 
blepharoplast  cannot  be  observed.  Another  more  advanced  stage  of 
multiple  fission  is  shown  in  the  somatella  in  figure  27.  The  number  of 
nuclei  and  of  the  parabasal  bodies  is  the  same  as  in  figure  26,  but  the 
outgrowths  of  the  daughter-flagella  are  clearly  shown  in  this  sphere. 
Spheres  without  protruding  flagella  are  to  be  found  in  figures  28  and 
29,  plate  3.  The  formation  of  the  merozoites  within  the  spheres,  as 
presented  in  figure  29,  has  advanced  to  the  point  where  they  are 
clearly  defined.  In  figure  28  another  important  observation  can  be 
made,  namely,  that  the  divisions  of  the  blepharoplast  and  the  nucleus 
do  not  occur  simultaneously.  In  this  particular  sphere  there  are  three 
parabasal  bodies  present  but  only  two  nuclei.  In  figure  29  there  are 
four  merozoites  visible,  each  of  which  shows  the  outgrowth  of  a  flag- 
ellum, and  a  similar  spherical  formation  is  shown  in  figure  30,  wherein 
the  four  merozoites  are  somewhat  larger  and  more  developed.  In  the 
latter  somatella,  however,  the  thickness  of  the  sphere  prevented  the 
usual  amount  of  destaining  necessary  to  show  the  nuclear  structure. 
In  each  of  these  merozoites  the  body  is  elongating  and  the  anterior 
end  is  becoming  attenuate. 

Another  thick  sphere  is  found  in  figure  31,  and  all  the  nuclei 
therein  have  the  appearance  of  being  a  solid  mass  of  chromatin.  In 
this  somatella  the  number  of  merozoites  which  could  be  counted  is 
twelve.  The  irregular  outline  of  the  sphere  indicates  that  the  breal\- 
ing  up  or  plasmotomy  of  the  sphere  is  about  to  occur.  Possibly  some 
of  the  merozoites  have  already  escaped.  In  the  investigation  thus 
far  the  number  of  merozoites  in  a  somatella  has  been  exceedingly 
variable.  In  some  of  the  Polymastigina,  Kofoid  and  Swezy  (1915) 
found  the  number  of  merozoites  to  be  eight,  which  is  apparently  con- 
stant for  the  somatellas  of  these  flagellates.  Minchin  and  Thomson 
(1915)  report  a  variable  number  of  merozoites  in  the  spheres  of 
Trypanosoma  lewisi  but  they  found  the  average  number  to  be  approxi- 
mately ten.  In  the  spherical  mass  of  flagellates  shown  in  figure  31, 
plate  3,  the  number  is  twelve,  but  in  a  still  larger  sphere  (pi.  3,  fig. 
32)  the  number  is  probably  double  that,  or  twenty-four.  The  dense- 
ness  of  the  latter  may  have  obscured  some  of  the  parabasal  bodies  and 
nuclei.  Protruding  from  the  surface  of  this  sphere  are  numerous  flag- 
ella which  are  outgrowths  from  the  daughter-blepharoplasts.  In  the 
somatellas  of  the  Polymastigina  the  nucleus  and  the  extranuclear 
organelles  may  divide  simultaneously,  but  in  the  spheres  of  T.  leivisi 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma        165 

and  in  the  somatella  of  Crithidia  euryophthalmi  either  the  extra- 
nuclear  or  the  nuclear  organelles  may  divide  first.  The  ultimate 
number  of  nuclei  is  equal  to  the  number  of  parabasal  bodies  and  of 
the  blepharoplasts. 

The  stage  of  development  of  the  merozoites  when  plasmotomy 
occurs,  is  variable.  Usually  the  rupture  of  the  somatella  occurs  when 
the  merozoites  are  just  beginning  to  elongate,  as  in  figure  31.  All 
the  organeles  of  these  merozoites  are  definitely  outlined,  and  the 
flagella  are  free  for  a  certain  distance  of  their  length.  In  a  few 
instances  extremely  elongate  flagellates  have  been  observed  wriggling 
about  within  the  spherical  structures.  There  is  no  evidence  of  a 
residual  mass  of  cytoplasm. 

A  comparison  of  these  two  methods  of  multiple  fission  in  the 
life  cycle  of  Crithidia  euryophthalmi  reveals  the  fact  that  they  are 
fundamentally  different,  In  the  first  place,  the  former  method,  endo- 
genous budding,  involves  only  one  organelle  of  the  crithidia,  namely, 
the  nucleus.  In  the  latter  method  a  somatella  results  from  a  repeated 
division  of  the  nucleus,  blepharoplast,  parabasal  body,  and,  in  all 
probability,  the  nuclear  and  parabasal  rhizoplasts.  The  flagella,  how- 
ever, in  each  case  are  new  outgrowths  from  the  newly  formed  blepharo- 
plasts in  endogenous  buds  or  from  the  daughter-blepharoplasts  in  the 
somatella.  Secondly,  the  flagellates  undergoing  endogenous  budding 
retain  their  normal  shape  as  elongate  or  pear-shaped  crithidias.  In 
some  instances  their  size  increases  as  the  buds  develop.  The  flagellates 
forming  somatellas  round  up  into  spheres.  The  comparative  efficiency 
of  the  two  processes  from  the  standpoint  of  the  multiplicative  phase 
in  the  host  cannot  be  estimated.  The  length  of  time  necessary 'to  com- 
plete each  process  and  the  conditions  under  which  each  occurs  are  at 
present  unknown.  On  the  whole,  the  number  of  individuals  resulting 
from  an  equal  number  of  endogenous  flagellates  or  of  somatellas  is 
approximately  the  same.  Our  conclusion,  however,  concerning  the 
two  methods  is  that  the  endogenous  budding  is  of  greater  importance 
in  the  life  cycle  of  C.  euryophthalmi  since  in  the  preparations  there 
are  relatively  many  more  evidences  of  the  endogenous  budding  than 
of  the  somatella. 

BINAEY  FISSION 

Another  method  of  multiplication  of  Crithidia  euryophthalmi  in 
the  "crop"  is  binary  fission.  This  process  has  also  been  observed  in 
the  pyloric  expansion.  While  the  number  of  crithidias  found  dividing 


166  University  of  California  Publications  in  Zoology       [VoL- 19 

in  this  way  is  relatively  small,  on  the  whole,  yet  crithidias  of  almost 
any  stage  of  development  apparently  may  thus  increase  their  number. 
Some  conception  of  the  prevalence  of  the  process  throughout  the 
life  cycle  may  be  gained  by  studying  figures  33  to  37.  In  addition 
to  these  smaller  crithidias  in  various  stages  of  development  many 
instances  of  binary  fission  have  been  observed  among  the  elongate  or 
mature  flagellates. 

The  blepharoplast  or  the  centrosome  of  the  nucleus  may  initiate 
the  division  of  the  several  organelles.  In  figure  33,  plate  3,  the 
centrosome  of  the  nucleus  is  in  the  process  of  binary  fission  and  there 
are  present  two  daughter-nuclei.  In  each  of  the  nuclei  the  chromatin 
is  peripheral  on  the  nuclear  membrane.  The  blepharoplast  and  the 
parabasal  body  have  not  yet  begun  to  divide.  The  general  appear- 
ance of  this  binary  fission  form  is  very  similar  to  that  of  the  smaller 
somatella.  In  the  unflagellated  crithidia  shown  in  figure  34,  plate  3, 
the  blepharoplast  and  parabasal  body  have  divided  but  the  nucleus 
is  still  intact.  A  more  advanced  stage  of  binary  fission  is  found  in 
figure  35.  In  this  small,  unflagellated  crithidia  the  blepharoplast, 
parabasal  body,  and  the  nucleus  have  divided,  and  a  light,  thin  area, 
which  is  preliminary  to  the  cleft  in  the  cytoplasm,  extends  longi- 
tudinally between  the  two  sets  of  organelles.  The  flagella  can  also 
be  observed.  The  longer,  more  clearly  defined  flagellum  is  evidently 
that  of  the  parent  since  the  second  is  shorter  and  less  distinct.  The 
last  stages  of  binary  fission  are  shown  in  figures  36  and  37.  In  figure 
36  the  cleft  in  the  cytoplasm  can  be  traced  from  the  anterior  to  the 
posterior  end,  and  the  daughter  flagellum  has  also  attained  a  greater 
length. '  The  two  flagella  in  figure  37  are  the  same  length;  the  separa- 
tion of  the  two  daughter  individuals  is  more  marked  and  almost  com- 
plete in  figure  38.  The  posterior  ends  are  the  last  to  remain  attached 
and  the  lashing  about  of  the  anterior  ends  assists  efficiently  in  the 
final  tearing  apart. 

One  of  the  most  interesting  problems  in  connection  with  this 
method  of  reproduction  has  always  been  the  origin  of  the  daughter- 
flagellum.  Is  it  due  to  the  division  of  the  flagellum  of  the  parent  or 
to  a  new  outgrowth  from  the  daughter-blepharoplast  ?  A  review 
of  the  work  already  done  on  this  genus  indicates  that  with  the  excep- 
tion of  Porter  (1909,  1910)  all  authors  regard  the  daughter-flagellum 
as  a  new  outgrowth  and  consider  that  the  parent-flagellum  does  not 
split  to  form  two  daughter  flagella.  My  work  on  Crithidia  leptocoridis 
and  C.  euryophthalmi,  unlike  that  of  Porter,  is  an  agreement  with 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma        167 

the  results  of  other  investigators.  For  some  time  the  evidence  of  the 
new  outgrowth  was  difficult  to  obtain  (McCulloch,  1915),  but  very 
clear  evidence  of  such  an  outgrowth  from  the  blepharoplast  has  finally 
been  discovered.  Text-figure  C  gives  a  very  clear  picture  of  the  origin 
of  the  daughter-flagellum  in  C.  leptocoridis.  The  larger  size  and  the 
greater  abundance  of  crithidias  undergoing  this  process  make  C.  lepto- 
coridis the  most  desirable  material  to  illustrate  this  point. 

The  importance  of  binary  fission  in  increasing  the  number  of 
parasites  in  the  multiplicative  phase  in  the  "crop"  is  not  great.  At 
the  present  time  no  evidence  of  the  process  has  been  found  in  the  mid- 
stomach,  which  serves  chiefly  as  a  passage  way  for  the  crithidias  mi- 


Fig.  C.  Flagellate  stage  of  Crithidia  leptocoridis  to  show  the  outgrowth  of 
the  new  flagellum  in  binary  fission.  X  3500.  Abbreviations  the  same  as  in 
figure  A. 

grating  from  the  "crop"  to  the  pyloric  expansion.  In  the  pyloric 
portion  of  the  digestive  tract,  however,  binary  fission  is  of  great  im- 
portance in  increasing  the  number  of  crithidias,  which  attach  them- 
selves to  the  epithelial  lining  of  the  pyloric  expansion. 

In  connection  with  the  process  of  binary  fission  in  Crithidia  eury- 
ophthalmi  another  problem  of  interest  has  occurred,  namely,  the  nature 
of  the  difference,  if  there  be  any,  between  the  division  of  the  several 
organelles  in  binary  fission  and  the  division  of  these  same  organelles  in 
the  somatella.  Minchin  and  Thomson  (1915)  have  regarded  the  in- 
crease in  the  number  of  nuclei  and  kinetonuclei  in  the  spheres  of  Try- 


168  University  of  California  Publications  in  Zoology       [VOL.  19 

panosoma  lewisi  as  being  due  to  a  repeated  binary  fission  wherein  the 
resulting  individuals  failed  to  separate  immediately.  Kofoid  and 
Swezy  (1915)  have  described  in  detail  the  process  of  binary  fission  and 
of  multiple  fission  for  the  trichomonad  flagellates,  and  these  authors 
found  the  increase  of  organelles  in  multiple  fission  to  be  due  to  thrice 
repeated  mitosis.  The  larger  size  of  those  flagellates  together  with 
the  correspondingly  increased  size  of  the  organelles  presents  better 
material  for  the  study  of  binary  and  multiple  fission  than  do  the 
trypanosomes  or  the  crithidias.  In  C.  euryophthalmi  the  minute  size 
of  the  flagellates  undergoing  either  process  have  made  accurate  inter- 
pretation thus  far  impossible.  For  this  reason  the  relation  of  binary 
fission  to  multiple  fission  in  a  somatella  must  remain  an  open  question 
for  the  present. 


INTRACELLULAR  CRITHIDIAS 

Another  salient  similarity  between  the  life  cycle  of  Crithidia 
euryophthalmi  and  the  life  cycle  of  Try  panosoma  lewisi  in  the  inverte- 
brate host  is  the  appearance  in  each  life  cycle  of  a  stage  of  intra- 
cellular  multiple  fission.  In  C.  euryophthalmi  there  is  figured  for 
the  first  time  an  epithelial  cell  in  the  life  cycle  of  a  crithidia  taken 
from  a  '"crop"  containing  numerous  crithidial  parasites.  Careful 
examination  of  this  infected  cell  shows  that  there  are  three  distinct 
groups  (pi.  3,  fig.  39,  a,  b,  c)  of  parasites  and  a  number  of  scattered 
crithidias.  Altogether  there  are  approximately  seventy  parasites  in 
this  one  epithelial  cell.  In  figure  39a  the  crithidias  are  of  two  sizes: 
long,  slender  flagellates,  and  short,  non-flagellated  forms.  Since  mul- 
tiple fission  of  either  the  endogenous  or  somatella  type  produces  zooids 
or  merozoites  of  approximately  the  same  size,  it  appears  that  several 
infections  have  occurred  in  this  cell.  It  is  conceivable  that  all  the 
elongate  flagellates  are  due  to  one  infection  while  the  short,  non-flag- 
ellated crithidias  are  due  to  a  second  infection.  In  figure  39&  there 
are  ten  small  oval  forms.  In  structure  they  show  a  diffuse  nucleus, 
as  do  the  other  parasites  within  this  host-cell,  which  is  probably  due 
in  part  to  the  thickness  of  the  cell.  A  nuclear  rhizoplast  can  be 
observed  passing  from  the  nucleus  to  the  blepharoplast,  and  a  short 
intracellular  flagellum  extends  forward  to  the  anterior  end  of  the 
body.  The  parabasal  body  of  each  is  a  relatively  small  and  deeply 
staining  structure.  At  c  and  e  of  figure  39  are  more  of  the  small  oval 
forms.  In  the  former  region  the  cytoplasm  of  the  host-cell  has  been 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma        169 

completely  destroyed  within  a  certain  radius  of  the  parasites.  In 
the  latter  the  small  forms  are  scattered  in  the  cytoplasm  of  the  host- 
cell.  At  d  there  is  still  another  group  of  elongate  flagellates,  which 
are  probably  the  results  of  another  process  of  multiple  fission.  They 
are  approximately  the  same  size  and  shape,  and  are  in  the  same  stage  of 
development.  Whether  they  are  merozoites  from  a  somatella  or  zooids 
from  endogenous  buds  is  not  clear,  but  they  have  broken  apart  some- 
what and  are  making  their  way  to  the  periphery  of  the  host-cell.  At  / 
a  flagellate  is  protruding  through  the  cell  wall  and  the  posterior  end 
is  directed  forward  to  penetrate  the  tissue.  In  the  study  of  the  living 
material  of  both  C.  leptocoridis  and  C.  euryophthalmi  crithidias  have 
been  observed  to  direct  their  posterior  ends  forward  and  to  use  the 
flagellated  ends  as  propellers  in  penetrating  tissues.  . 

The  early  stages  of  the  intracellular  multiple  fission  have  not  yet 
been  found  within  the  epithelial  cells  of  the  crop  and  the  more  ad- 
vanced stages,  such  as  shown  in  figure  39,  do  not  indicate  definitely  the 
method  of  multiple  fission.  These  intracellular  crithidias  had  been 
described  as  the  results  of  multiple  fission  within  somatellas  before 
the  discovery  of  the  stages  of  the  endogenous  process  of  multiple 
fission  in  this  life  cycle.  The  evidence  is  not  absolutely  convincing 
either  way,  but  there  is  at  the  present  state  of  the  investigation  a 
preponderance  of  evidence  in  favor  of  their  probable  origin  by  the 
plasmotomy  of  a  somatella.  The  circular  outlines  of  the  vacuoles  in 
which  the  groups  of  parasites  are  found  in  figure  39  suggest  the 
formation  of  the  spheroidal  somatella.  Further  there  are  no  evi- 
dences of  discarded  flagella  within  these  cells  which  suggests  the  possi- 
bility that  endogenous  budding  might  have  occurred  with  the  forma- 
tion of  circular  cavities  in  the  cytoplasm  of  the  host-cell.  On  the 
other  hand,  if  the  numerous  small  oval  spores  are  due  to  a  process  of 
multiple  fission  within  a  somatella  the  subsequent  plasmotomy  has 
taken  place  very  early.  Usually  the  breaking  apart  of  the  merozoites 
does  not  occur  until  they  have  become  elongate  flagellates.  Moreover, 
the  exact  method  of  the  process  of  intracellular  multiple  fission  is 
not  so  important  as  the  fact  that  under  certain  conditions  crithidias 
become  intracellular  and  destroy  the  epithelial  lining  of  the  digestive 
tract  which  they  penetrate.  Each  destruction  of  a  host-cell  thus  also 
means  a  tremendous  increase  apparently  in  the  number  of  the  para- 
sites. The  intracellular  crithidias  are  not  found  frequently  in  the 
preparations.  We  have  no  proof  that  it  is  an  obligatory  phase,  though 
it  might  well  be  so.  Nor  have  we  evidence  that  it  follows  the  forma- 


170  University  of  California  Publications  in  Zoology       [VOL.  19 

tion  of  a  zygote,  as  does  the  somatella  of  Plasmodium  in  the  wall  of 
the  digestive  tract  of  a  mosquito.  There  is  no  evidence  that  the  zygote 
precedes  the  somatella  in  the  Polymastigina.  In  the  life  cycle  of 
Trypanosoma  lewisi  the  process  of  intracellular  multiple  fission  is 
apparently  obligatory.  The  trypanosomes,  or  haemoflagellates,  pene- 
trate epithelial  cells  of  the  "crop,"  undergo  multiple  fission,  and 
crithidiomorphic  merozoites  are  produced.  This  phase  brings  about  the 
early  stages  of  transition  from  a  trypanosome  to  a  crithidia.  A  similar 
need  for  such  a  phase  is  not  present  in  the  life  cycle  of  Crithidia 
euryophthalmi.  At  present  our  knowledge  of  intracellular  multiple 
fission  in  the  life  cycle  of  C.  euryophthalmi  is  too  meager  to  permit  of 
extended  correlation  between  the  life  cycle  of  this  more  primitive 
flagellate  with  that  of  the  more  highly  developed  haemoflagellate  T. 
leivisi.  It  is  conceivable  that  in  the  evolution  of  trypanosomes  from 
the  crithidial-like  flagellates  the  intracellular  multiple  fission  was 
carried  over  and  became  more  specialized  and  more  important  in  the 
life  cycle  of  the  haemoflagellate. 


RECTAL  PHASE 

The  stomach  phase  of  Crithidia  euryophthalmi,  beginning  with  the 
initial  infective  spores  and  ending  with  the  great  swarm  of  parasites 
resulting  from  binary,  extracellular,  and  intracellular  multiple  fission 
in  the  "crop,"  is  followed  by  the  established  rectal  phase  of  the  life 
cycle  in  the  pyloric  expansion. 

Owing  to  the  structure  of  the  digestive  tract  of  Euryophthalmus 
convivus,  with  its  three  divisions  separated  only  by  a  narrow  con- 
striction, which  allows  a  possible  intermingling  under  normal  condi- 
tions of  the  crithidias  in  the  "crop"  with  those  of  the  mid-stomach 
and  pyloric  expansion,  it  is  extremely  difficult  to  say  where  the  stomach 
phase  ends  and  the  rectal  phase  begins.  In  Trypanosoma  lewisi  the 
transition  between  the  stomach  and  rectal  phase,  as  has  already  been 
pointed  out,  is  marked  by  definite  structural  changes.  The  trypano- 
somes, brought  into  the  stomach  of  the  flea  with  blood  from  a  rat,  enter 
epithelial  cells  and  undergo  multiple  fission,  producing  merozoites  of 
a  crithidiomorphic  type.  The  merozoites  thus  produced  may  do  one 
of  two  things,  either  enter  other  epithelial  cells  of  the  stomach  or 
collect  at  the  pyloric  opening  and  be  carried  down  the  intestine  to  the 
rectum  with  food.  As  they  pass  through  the  intestine  the  structural 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma        171 

changes  which  convert  crithidiomorphic  forms  into  crithidias  are 
taking  place. 

The  frequency  with  which  we  find  preparations  of  the  "crop" 
free  from  infection  with  C.  euryophthalmi,  the  less  frequency  of  infec- 
tion of  the  mid-stomach,  together  with  the  almost  invariably  heavy 
infection  in  the  pyloric  expansion,  lead  us  to  the  conclusion  that  the 
crithidias  of  Euryophthalmus  convives  have  the  same  tendency  to 
migrate  posteriorly  as  does  T.  lewisi  in  the  flea.  In  C.  euryophthalmi, 
however,  the  movement  is  less  marked  than  in  T.  lewisi.  This  tendency 
of  slow  progression  posteriorly  doubtless  depends  upon  the  move- 
ment of  food  contents  from  the  "crop"  into  the  mid-stomach  and 
pyloric  expansion. 

The  migrating  mass  of  crithidias  from  the  '  *  crop ' '  soon  establishes 
three  distinct  types  of  parasites  in  the  pyloric  expansion:  the  necto- 
monads  or  free  flagellates,  the  haptomonads  or  attached  flagellates, 
and  the  infective  spores  which  serve  for  transmitting  C.  euryophthalmi 
to  another  host.  These  types  or  classes  of  parasites  are  comparable  in 
almost  every  way  to  the  nectomonads,  or  free  flagellates,  the  hapto- 
monads, or  attached  flagellates,  and  the  final  trypaniform  stage  of  the 
rectal  phase  of  T.  lewisi.  The  nectomonads  and  haptomonads  of  each 
life-cvcle  are  almost  identical  in  structure  and  behavior. 


NECTOMONADS 

Smear  preparations  of  the  mid-stomach  and  of  the  pyloric  expan- 
sion show  little  difference  in  the  morphological  structure  of  their 
crithidial  infection.  The  serial  sections  of  these  two  parts,  however, 
show  a  sharp  distinction  in  the  structure  of  haptomonad  forms  and 
the  nature  of  the  epithelial  lining  to  which  they  are  attached,  and 
a  slight  distinction  in  the  structure  of  the  nectomonads  in  the  mid- 
stomach  and  in  the  pyloric  expansion.  The  nectomonads  of  the 
former  are  usually  of  the  elongate,  slender  type  (pi.  5,  figs.  73-80). 
The  zooids,  the  result  of  the  processes  of  multiple  fission  (pi.  4, 
figs.  40-54)  are  abundant  in  the  smear  preparations  of  both  regions. 
The  series  sections  thus  far  have  shown  numerous  zooids  (pi.  4, 
figs.  40-50)  in  the  anterior  portion  of  the  mid-stomach.  These 
zooids  (pi.  4,  fig.  40)  are  frequently  grouped  together  in  the  grooves 
between  epithelial  cells.  For  this  reason  it  is  possible  that  the  cur- 
rent of  food  in  passing  down  the  digestive  tract  failed  to  carry 
them  on  into  the  pyloric  expansion.  The  zooids  are  small  forms, 


172  University  of  California  Publications  in  Zoology       [VOL.  19 

(pi.  4,  figs.  40-47)  from  I/*  (pi.  4,  fig.  40)  to  1.5ft  (pi.  4,  fig.  47)  in 
length  and  from  0.7  to  I/A  in  diameter.  In  comparing  them  with 
the  initial  infective  oval  spores  (pi.  2,  figs.  1,  2)  it  is  noticed  that  they 
are  smaller,  stain  less  densely,  and  do  not  show  a  heavy  periplast.  The 
nuclear  structure  also  is  unlike  that  of  the  initial  spore  forms,  and  the 
location  of  the  nucleus  within  the  zooid  adds  another  distinguishing 
character.  These  zooids  have  always  been  found  in  the  anterior  por- 
tion of  the  mid-stomach  in  the  serial  sections  while  the  sections  imme- 
diately posterior  contain  developing  crithidias  (pi.  4,  figs.  59-64). 
Groups  of  elongating  forms  are  frequently  found  (pi.  4,  fig.  59). 
With  the  elongation  of  the  body  of  the  zooids,  especially  of  the  ante- 
rior end,  the  nucleus  instead  of  being  filled  with  chromatin  is  now 
vesicular  and  contains  a  distinct  karyosome.  This  group  (pi.  4, 
fig.  59)  shows  the  parabasal  bodies  in  close  proximity  to  the  nuclear 
membranes.  Judging  from  the  conditions  found  within  the  great 
majority  of  forms,  figure  59  is  probably  an  exceptional  case  in  this 
respect.  The  parabasal  body  normally  moves  anteriorly  in  the  early 
development  of  the  zooid  (pi.  6,  fig.  40)  before  the  flagellum  and  the 
anterior  end  grow  out  (pi.  4,  figs.  41-53).  In  the  figures  just  noted 
the  flagellum'  is  not  yet  visible.  The  nuclear  rhizoplast,  extending 
from  the  nucleus  forward  to  the  region  of  the  blepharoplast  and  para- 
basal body,  is  found  by  focusing  carefully.  In  figure  54  the  flagellum 
is  growing  out  from  the  blepharoplast  but  there  is  no  noticeable 
lengthening  of  the  anterior  end  of  the  body.  In  figures  55  to  58  the 
flagellum  and  anterior  end  of  each  are  lengthening  simultaneously. 
In  these  same  figures  the  nuclei  are  like  the  nuclei  of  the  zooids,  being 
completely  filled  with  chromatin.  Farther  on  posteriorly  are  found 
crithidias  such  as  are  shown  in  figures  63  to  72.  Beginning  with 
figure  65  there  is  also  an  elongation  of  the  posterior  end,  which  is  equal 
to  that  of  the  anterior. 

The  majority  of  these  developing  crithidias  have  the  vesicular 
type  of  nucleus.  Figures  65,  67,  and  68  are  exceptions,  but  no  sig- 
nificance can  be  attached  to  them  since  the  position,  relative  thickness 
of  the  body,  or  the  technique,  could  explain  these  exceptions  in  this 
region  of  the  digestive  tract.  The  study  of  the  serial  sections  leads 
us  to  think  that  the  time  necessary  for  the  stomach  crithidias  to 
reach  the  rectum  is  approximately  the  amount  of  time  required  for 
the  zooids  to  develop  into  mature  flagellates.  Not  all  of  the  develop- 
ing zooids  become  mature  in  the  mid-stomach.  Under  certain  con- 
ditions the  food  current  probably  carries  many  of  the  non-flagelated 
stages  or  zooids  back  into  the  pyloric  expansion  before  they  have 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma        173 

scarcely  begun  to  develop.  As  the  developing  forms  and  the  fully 
matured  forms  enter  the  pyloric  expansion,  they  become  part  of  the 
permanent  crithidial  infection  therein.  The  content  of  the  crithidial 
infection  of  this  region  varies  from  time  to  time.-  Nectomonads 
may  predominate  or  all  nectomonads  may  become  haptomonads,  or 
attached  forms.  Usually  the  nectomonads  and  haptomonads  are  both 
present  in  large  numbers.  Mature  nectomonads  in  the  mid-stomach 
are  usually  the  elongate,  slender  crithidias  (pi.  4,  figs.  73-79).  In 
figure  74  the  body  size  is  approximately  25/x  in  length  and  1.7/*  in 
width.  Figures  75  to  78  are  yet  narrower,  averaging  about  I/A  in 
width.  The  nectomonads  of  the  pyloric  expansion  are  frequently 
like  figures  73  to  79,  long,  slender  forms  together  with  numerous 
shorter  and  stouter  erithidias  (pi.  4,  figs.  80-90).  It  is  conceivable 
that  the  elongate  slender  forms  give  rise  to  the  shorter,  stout  forms. 
The  length  of  the  body  is  decreased  and  the  width  is  increased.  Both 
the  anterior  and  the  posterior  ends  of  the  body  become  less  attenuate. 
The  structure  of  the  nucleus  of  the  elongate  forms  varies  con- 
siderably. In  figures  73  and  80  there  are  distinct  central  karyosomes 
with  chromatin-encrusted  membranes.  In  figure  74  the  chromatin 
is  in  five  granules  scattered  within  the  nuclear  membrane.  The 
chromatin  is  broken  up  into  granules  in  figures  76  and  79.  The  break- 
ing up  of  the  chromatin  material  in  these  nectomonad  forms  doubtless 
means  the  beginning  of  a  degeneration,  which  will  be  discussed  shortly. 
The  nuclear  structures  in  figures  77  and  78  are  unique.  In  figure 
77  the  nuclear  membrane  is  rather  densely  encrusted  with  chromatin 
and  is  elongate  and  irregular  in  outline.  In  figure  78  the  nuclear 
membrane  is  oval,  chromatin-encrusted,  with  two  masses  of  chromatin 
at  the  anterior  and  posterior  regions  of  the  nucleus.  In  the  series 
of  figures  81,  83  to  86  a  karyosome  of  a  variable  size  is  found  in 
each  and  its  location  is  not  always  central.  In  figure  82  a  distinct 
chromidial  fragmentation  of  the  chromatin  has  taken  place,  which  is 
another  indication  of  degeneration.  The  short,  stout  forms  (pi.  4, 
figs.  87-90)  are  transition  forms  from  the  nectomonads  to  hapto- 
monads. While  these  flagellates  are  still  free  forms  they  resemble  the 
haptomonads  of  the  pyloric  expansion,  which  are  attached  in  the  mid- 
region  of  this  division.  In  the  anterior  part  of  the  pyloric  expansion 
the  haptomonads  are  relatively  long,  slender  flagellates  while  posterior 
to  the  middle  portion  they  are  still  short  and  more  pear-shaped.  The 
nuclear  structure  of  these  transition  forms  indicates  no  degeneration 
as  yet.  They  all  have  a  central  karyosome  and  a  more  or  less  encrusted 
nuclear  membrane. 


174  University  of  California  Publications  in  Zoology       [VOL.  19 


HAPTOMONADS 

One  of  the  most  characteristic  features  of  the  life  cycle  of  a  crithi- 
dial  flagellate  is  the  great  mass  of  attached  forms  which  line  definite 
parts  of  the  digestive  tract  of  the  host.  Minchin  and  Thomson  found 
three  regions  of  the  digestive  tract  of  the  rat-flea  where  the  crithidias 
of  Trypanosoma  lewisi  might  attach  themselves,  namely,  the  prepyloric, 
post-pyloric,  and  the  rectal  regions.  In  the  lupine  bug  there  are  like- 
wise three  regions  where  the  haptomonads  may  attach  themselves :  ( 1 ) 
in  the  posterior  part  of  the  "crop,"  where  they  are  possibly  com- 
parable to  the  prepyloric  crithidias  in  the  posterior  part  of  the 
stomach  of  the  flea;  (2)  in  the  posterior  half  of  the  mid-stomach, 
where  they  are  probably  comparable  to  the  post-pyloric  crithidias 
of  the  rat-flea,  attached  to  the  anterior  part  of  the  intestine;  and  (3) 
in  the  pyloric  expansion,  where  they  are  comparable  to  the  hapto- 
monads of  the  rectum  of  the  rat-flea.  In  Trypanosoma  lewisi  in  the 
flea  these  investigators  regard  the  prepyloric  haptomonads  as  being 
due  to  a  forward  migration  from  the  rectum,. possibly  as  a  result  of 
the  food  conditions.  The  prepyloric  haptomonads  are  not  found  fre- 
quently in  the  flea,  and  in  only  one  preparation  of  the  "crop"  of  the 
lupine  bug  were  haptomonads  found.  In  the  lupine  bug  haptomonads 
were  found  in  a  number  of  preparations  of  the  mid-stomach,  but  they 
were  commonly  present  in  preparations  of  the  pyloric  expansion.  The 
haptomonads  observed  in  the  single  preparation  of  the  "crop"  were 
of  the  rectal  type,  small  oval  forms  similar  to  those  from  the  pyloric 
expansion  shown  in  plate  6,  figures  107  and  117.  The  serial  sections  of 
the  digestive  tract,  however,  showed  no  haptomonads  in  the  "crop." 

From  the  serial  sections  of  the  mid-stomach  abundant  material 
was  obtained  for  the  study  of  the  haptomonad  of  this  region.  The 
attached  forms  here  (pi.  6,  figs.  93-96)  are  relatively  small,  slender 
flagellates.  They  are  uniform  in  size  and  shape,  on  the  whole,  and 
form  a  definite  fringe  on  the  inner  surface  of  the  epithelial  lining  of 
the  sections  of  the  mid-stomach.  These  flagellates  attach  themselves 
to  the  epithelial  cells  by  means  of  the  flagella.  They  frequently  almost 
surround  the  elongate,  columnar  epithelial  cells,  which  project  into 
the  lumen  of  the  digestive  tract.  The  grooves  between  masses  of  epi- 
thelial cells  evidently  afford  a  particularly  good  place  for  the  hapto- 
monads, since  they  are  found  in  compact  layers  in  such  places.  Hapto- 
monad crithidias  of  this  type  are  the  only  ones  found  in  the  mid- 
stomach  and  they  continue  to  line  the  digestive  tract  posteriorly 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma        175 

into  the  pyloric  expansion.  Considering  the  attached  crithidias  the 
most  anterior  sections  of  the  pyloric  expansion  are  almost  identical 
with  the  sections  from  the  posterior  part  of  the  mid-stomach.  The 
lumen  of  the  narrow  constriction  separating  the  mid-stomach  from 
the  pyloric  expansion  is  frequently  almost  blocked  with  the  bodies  of 
the  flagellates  extending  out  into  the  passageway  between  these  two 
enlargements  of  the  digestive  tract. 

The  structure  of  these  haptomonads  (pi.  6,  figs.  93-96)  is  also 
relatively  uniform.  The  majority  of  the  crithidias  show  the  vesicular 
type  of  nucleus,  with  a  central  karyosome  and  a  nuclear  membrane 
containing  an  initer  lining  of  chromatin  material.  In  figure  94  the 
nuclear  membrane  is  slightly  distorted  in  appearance  and  the  karyo- 
some is  excentric,  being  posterior.  In  figure  96  the  nuclear  membrane 
is  not  distinct  and  the  enlarged  mass  of  chromatin  material  is  in  the 
form  of  two  granules.  These  haptomonads  are  usually  characterized 
by  their  attenuate  anterior  and  posterior  ends.  The  posterior  ends  of 
these  crithidias  more  than  of  any  others  show  relatively  extreme 
attenuation. 

Beginning  in  a  region  just  posterior  to  the  mid-stomach  end  of 
the  pyloric  expansion  a  series  of  transition  haptomonads  is  found 
(pi.  6,  figs.  97-106),  lining  the  middle  part  of  this  division  of  the 
digestive  tract,  these  transition  haptomonads,  which  vary  consider- 
ably in  size.  Figure  97  shows  a  broad,  stout  form.  Such  forms  undergo 
binary  fission,  producing  two  smaller,  more  slender  individuals.  Pos- 
sibly figures  100  and  101  are  the  products  of  such  a  division.  The 
binary  fission  of  the  broad,  stout  forms  rapidly  increases  the  number 
of  haptomonads  which  make  up  the  dense  and  compact  layer  of  attached 
crithidias.  The  nuclear  structure  of  these  forms  shows  compara- 
tively little  variation.  They  have  the  vesicular  nucleus  with  the  small 
central  karyosome  and  chromatin-encrusted  nuclear  membrane. 

At  the  posterior  portion  of  the  pyloric  expansion  are  found  the 
normal  rectal  forms  (pi.  6,  figs.  107-124)  which  are  common  to  the 
life  cycles  of  so  many  of  these  flagellates.  One  of  the  interesting 
things  observed  in  connection  with  these  forms  is  that  the  wall  of  the 
pyloric  expansion  becomes  exceedingly  thin.  There  are  few  indica- 
tions left  of  the  epithelial  cells  lining  this  part  of  the  mid-gut.  Sec- 
tions of  this  portion  of  the  tract  previous  to  any  infection  by  the 
flagellate  are  not  at  hand,  unfortunately,  and  consequently  it  is  diffi- 
cult to  estimate  the  total  amount  of  destruction  incurred.  The  pyloric 
expansion  of  an  infected  adult  bug,  however,  is  extremely  weak  and 


176  University  of  California  Publications  in  Zoology       [VOL.  19 

becomes  torn  very  readily ;  it  has  every  appearance  of  having  been 
almost  entirely  destroyed  by  the  crithidias.  Among  these  small  hapto- 
monads  numerous  variations  of  size  and  shape  are  noted.  The  length 
of  the  free  flagellum  (pi.  6,  figs.  109,  110)  is  exceedingly  variable. 
In  other  crithidias  whatever  flagella  are  present  are  intracellular 
throughout  their  length.  The  changes  brought  about  in  the  flagella 
are  probably  due  to  their  absorption.  In  every  case  the  flagellum 
attaches  the  crithidia  to  the  wall  of  the  digestive  tract.  The  nuclear 
structure  of  the  haptomonads  in  this  region,  with  the  exception  of 
the  forms  in  figure  91,  shows  no  indication  of  degeneration.  The  round 
haptomonads  finally  become  free  forms.  They  can  be  seen  to  drop 
off  in  the  living  preparations  and  to  roll  up  into  round  or  oval  forms 
(pi.  6,  figs.  118,  119,  122).  The  cytoplasm  of  these  forms  is  vacuolate 
and  stains  lightly.  These  round  crithidias  then  degenerate  along  with 
the  nectomonads,  which  are  constantly  degenerating.  The  degenera- 
tion of  the  haptomonads  and  nectomonads  will  be  described  under 
the  degenerative  series. 

FINAL  SPOEE  FORMS 

The  structure  of  the  digestive  tract  of  EuryophtJialmus  co-nvivus, 
including  three  portions  of  the  stomach  proper  and  the  intestine  with 
its  gland,  differentiates  very  clearly  between  the  degenerative  series 
of  crithidias  and  the  final  spore  forms  which  can  be  transmitted  to 
another  host.  In  the  anterior  part  of  the  digestive  tract  is  found  the 
developmental  series  which  becomes  the  degenerative  series  together 
with  the  final  spore  forms.  Posterior  to  the  gland  only  the  final 
spore  forms  have  been  observed.  The  preparations  of  the  rectum 
show  only  these  final  spore  forms  (pi.  6,  figs.  125-131).  These  are 
oval,  non-flagellated  forms  containing  a  thick  periplast,  within  which 
are  the  nucleus  and  parabasal  body.  In  figure  126  the  characteris- 
tics of  these  final  spore  forms  may  be  noted.  They  are  approximately 
2.8/>t  in  length  and  1.4ft  in  width.  The  nucleus  lies  in  the  extreme 
posterior  end  of  the  body  and  stains  deeply.  The  parabasal  body  is 
sharply  outlined  within  a  vacuolate  area.  A  faint  nuclear  rhizoplast 
may  be  visible,  extending  from  the  nucleus  toward  the  parabasal 
body.  As  previously  indicated  in  a  preliminary  paper  (McCulloch, 
1917)  it  was  some  time  before  the  true  rectum  was  discovered,  and 
consequently  the  significance  of  these  final  spore  forms  was  not  en- 
tirely clear  in  the  beginning  of  the  investigation.  However,  with  the 
discovery  of  the  true  rectum  and  the  fact  that  it  contained  only  these 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma        177 

final  spores  the  characteristics  of  these  rectal  forms  became  clear  and 
it  was  relatively  easy  to  find  them  in  both  the  mid-stomach  and  pyloric 
expansion.  In  the  "crop"  of  the  lupine  bug  the  multiple  fission 
gives  rise  to  numerous  small  zooids.  Some  few  of  these  zooids,  on 
reaching  a  certain  stage  of  development,  become  the  well  protected 
final  spore  forms.  Their  development  into  mature  flagellates  is  ar- 
rested for  an  indefinite  time,  perhaps  in  response  to  some  unfavorable 
internal  or  external  chemical  condition.  The  periplast  becomes  thick- 
ened and  these  zooids  stain  deeply  and  retain  the  stain  much  better 
than  the  unprotected  zooids.  Since  only  a  few  of  the  zooids  be- 
come thus  encysted  the  change  cannot  be  regarded  as  a  general 
change  in  response  to  some  general  stimulus.  In  the  infections  of 
the  mid-stomach  and  pyloric  expansion  some  of  the  final  spore  forms 
can  be  found  at  almost  any  period  in  the  life  history  of  the  flagellates. 
Considering  that  these  are  the  only  stage  of  the  life  cycle  of  the 
flagellate  which  have  been  found  in  the  rectum  they  have  been  re- 
garded as  the  final  spore  forms,  which  upon  being  ingested  with  food 
by  another  insect  host  become  the  initial  infective  spores.  Our  observa- 
tions upon  these  cannot  be  regarded  as  conclusive  as  yet,  owing  to 
the  fact  that  experimentally  we  have  not  produced  infection  with 
these  spores. 

THE  DEGENERATIVE  SERIES 

The  degenerative  series  includes  all  individuals  of  the  life  cycle 
other  than  the  final  spore  forms  and  their  antecedents  which  have 
just  been  discussed.  It  has  already  been  pointed  out  that  the  rectal 
phase  of  C.  euryophthalmi  in  the  pyloric  expansion  of  the  lupine  bug 
is  comparable  to  the  rectal  phase  of  T.  lewisi  in  the  rectum  of  the 
flea.  In  the  lupine  bug  the  degenerating  forms  abound  in  the  poste- 
rior portion  of  the  pyloric  expansion  and  their  number  is  apparently 
not  decreased  by  constant  elimination  under  normal  conditions  of 
such  parasites  from  the  intestinal  contents,  which  pass  through  a 
relatively  long  intestine  before  reaching  the  colon  and  rectum.  If 
any  flagellates  succeed  in  passing  out  with  the  intestinal  matter  into 
this  long  intestine  they  are  evidently  destroyed  by  the  new  chemical 
medium  before  they  reach  the  rectum.  In  the  flea  the  degenerating 
forms  are  in  the  rectum  and  it  is  possible  that  their  number  is 
repeatedly  being  decreased  with  each  discharge  of  the  feces. 

There  is  little  danger  of  confusing  the  developmental  and  degenera- 
tive series  in  the  life-cycle  of  C.  euryopkthalmi  since  the  differentia- 


178  University  of  California  Publications  in  Zoology       [VOL.  19 

tion  of  the  parasites  into  the  final  spore  forms  and  the  ordinary 
propagative  forms  takes  place  apparently  in  the  stomach  phase  in 
the  *  *  crop. ' '  The  majority,  in  fact,  nearly  all  of  the  parasites  become 
ordinary  propagative  forms  which  develop  and  increase  their  num- 
bers in  the  digestive  tract  of  the  lupine  bug.  After  passing  through 
the  various  processes  of  the  life  cycle  they  degenerate  in  the  late 
rectal  phase  in  the  pyloric  expansion.  The  permanent  rectal  phase 
persists  through  the  life  of  the  lupine  bug  after  the  first  infection; 
the  degenerative  series  is  soon  formed,  and  likewise  persists  throughout 
the  life  of  the  host. 

The  individuals  of  the  degenerative  series  are  distinguished  among 
the  mass  of  living  crithidias  by  a  sticky  periplast,  to  which  bacteria 
frequently  adhere  by  virtue  of  the  tendency  of  crithidias  to  adhere  to 
each  other,  by  slow  sluggish  movements,  and  by  odd  sizes  and  shapes. 
The  degenerating  forms  are  detected  in  stained  preparations  by  nuclei 
with  diffused  chromatin  or  by  a  vesicular  nucleus  breaking  up  into  a 
number  of  chromatin  granules,  and  by  the  vacuolated  cytoplasm.  The 
size,  shape,  and  location  of  the  crithidias  also  assists  in  distinguishing 
between  the  developmental  and  degenerative  crithidias. 


CONCLUSIONS 

1.  The  crithidial  flagellates  of  the  life  cycle  of  Trypanosoma  are 
structurally  like  the  crithidial  flagellates  of  the  life  cycle  of  Crithidia. 
The  extranuclear  organelles,  the  blepharoplast,  parabasal  body,  para- 
basal  rhizoplast,  nuclear  rhizoplast,  and  the  flagellum  are  all  common 
to  the  crithidial  flagellates  of  both  Trypanosoma  and  Crithidia. 

2.  From  the  viewpoint  of  comparative  morphology  the  differences 
existing  between  the  crithidial  forms  of  C.  euryophthalmi  and  the 
crithidial  forms  of  T.  lewisi  are  less  marked  than  are  the  differences 
between  similar  stages  of  T.  lewisi  and  Schizotrypanum  cruzi. 

3.  Using  the  life  cycle  of  T.  lewisi  as  a  standard  for  comparison  of 
the  life  cycle  of  a  haemoflagellate  or  a  trypanosome  and  the  life  cycle 
of  C.  euryophthalmi  as  the  standard  of  the  life  cycle  of  a  more  primi- 
tive crithidial  flagellate,  there  are  more  parallel  stages  and  phases  in 
these  two  life  cycles  than  exist  between  the  life  cycle  of  any  trypano- 
some and  the  life  cycle  of  any  herpetomonad  or  of  any  leptomonad  now 
known.     Furthermore  the  close  correlation  between  these  two  life 
cycles  of  T.  lewisi  and  of  C.  euryophthalmi  affords  new  evidence  that 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma        179 

the  evolution  of  a  trypanosome  has  probably  taken  place  from  a 
crithidial  flagellate  rather  than  from  a  herpetomonad  or  leptomonad 
flagellate. 

4.  The  process  of  multiple  fission  in  the  somatella  of  C.  euryoph- 
thalmi  is  fundamentally  like  the  multiple  fission  (sphere  formation) 
of  T.  lewisi  and  also  like  the  multiple  fission  (somatella)  of  the  tricho- 
monad  flagellates.    In  each  of  these  flagellates  the  nucleus,  parabasal 
body,  blepharoplast,  and  flagellum,  or  flagella,  are  concerned  in  the 
process.     In  each  after  multiple  fission  plasmotomy  occurs. 

5.  The  process  of  multiple  fission  by  endogenous  budding  in  the 
life-cycle  of  C.  euryophthalmi  tends  not  only  to  establish  another  link 
in  common  between  the  life  cycles  of  Trypanosoma  (e.g.,  T.  gambiense) 
and  the  life  cycle  of  Crithidia  but  also  to  link  the  life  cycle  of 
Crithidia  more  closely  to  the  lower  protozoan  forms  which  contain 
numerous  Leishmania-like  bodies  in  their  life  cycles. 

6.  The  endogenous  buds  in  the  life  of  C.  euryophhtalmi  are  com- 
parable to  the  latent  bodies  in  the  life-cycle  of  T.  gambiense. 


180  University  of  California  Publications  in  Zoology       [VOL.  19 


LITERATURE  CITED 

CHATTON,  E.,  and  LEGER,  A. 

1911.     Eutrypanosomes,    Leptomonas  et    d'un    Trypanosoma   et   leptotrypano- 
somes  chez  Drosophila  confusa  (Muscide).     C.  E.   Soc.  Biol.  Paris, 
70,  34-36. 
CHAGAS,  C. 

1909.  Ueber  eine  neue  Trypanosomiasis  des  Menschen.  Mem.  Tnst.  Osw. 
Cruz,  1,  159-218,  pis.  9-13,  10  figs,  in  text. 

FANTHAM,  H.  B.,  and  PORTER,  A. 

1915a.  Further  experimental  researches  on  insect  flagellates  introduced  into 

vertebrates.     Proc.  Cambridge.    Philos.  Soc.,  18,  137-148. 
1915b.  On  the  natural  occurrence  of  herpetomonads  (leptomonads)  in  mice. 

Parasitology,  8,  128-132,  7  figs,  in  text. 
1915c.  Some  experimental  researches  on  induced  herpetomoniasis  in  birds. 

Ann.  Trop.  Med.,  9,  543-558,  pi.  39. 
FRANQA,  C. 

1914.  La  flagellose   des  Euphorbes.     Arch.  f.   Prot.,   34,   108-132,   pi.   5,   4 

figs,  in  text. 

HARTMANN,  M. 

1911.  Die   Konstitution   der   Protistenkernei     Jena,   Fischer,  pp.    1-54,   13 

figs,  in  text. 
KOFOID,  C.  A. 

1916.  The  biological  and  medical  significance  of  the  life-history  of  intes- 
tinal flagellates.  Proc.  Sec.  Pan-Amer.  Sci.  Cong.,  Washington, 
1915-16,  10,  546-565. 

KOFOID,  C.  A.,  and  McCuLLOCH,  I. 

1916.  On  Trypanosoma  triatomae,  a  new  flagellate  from  a  hemipteran  bug 

from  the  nests  of  the  wood  rat,  Neotoma  fuscipes.     Univ.  Calif. 
Publ.  Zool.,   16,   113-126,  pis.   14-15. 

KOFOID,  C.  A.,  and  SWEZY,  O. 

1915.  Mitosis  and  multiple  fission  in  trichomonad  flagellates.     Proc.  Am. 

Acad.  Arts  and  Sci.,  51,  287-379,  pis.  1-8,  7  figs,  in  text. 

MCCULLOCH,   I. 

1915.  An  outline  of  the  morphology  and  life  history  of  Crithidia  leptocori- 
dis  sp.  nov.  Univ.  Calif.  Publ.  Zool.,  16,  1-22,  pis.  1-4,  1  fig. 
in  text. 

1917.  Crithidia   euryophthalmi,  sp.   nov.,  from  the  hemipteran  bug,  Euryo- 

phthamus  convivus,  Stal.    Ibid.,  18,  75-88,  35  figs,  in  text. 

MINCHIN,  E.  A. 

1908.  Investigations  on  the  development  of  trypanosomes  in  tse-tse  flies 
and  other  Diptera.  Quart.  Jour.  Micr.  Sci.,  52,  159-260,  pis.  8-13-, 
2  figg.  in  text. 

1912.  An    introduction    to    the    study    of    Protoza.      London,    Arnold,    pp. 

1-520,  194  figs,  in  text. 


1919]      McCulloch:  Life  Cycle  of  Crithidia  and  Trypanosoma        181 

MINCHIN,  E.  A.,  and  THOMSON,  J.  D. 

1915.  The  rat-trypanosome,  Trypanosoma  lewisi,  in  its  relation  to  the  rat- 

flea,  Ceratophyllus  fasciatus.     Quart.  Jour.  Micr.  Sci.,  60,  463-692, 
pis.  36-45,  24  figs,  in  text. 

MOORE,  J.  E.  S.,  and  BREINL,  A. 

1907.  Cytology  of  the  trypanosomes,  I.     Ann.  Trop.  Med.,  1,  441-480,  pis. 

38-42. 

PATTON,  W.  S. 

1908.  Herpetomonas  lygaei.     Arch.  f.  Prot.,  13,  1-18,  pi.  1,  2  figs,  in  text. 

1908.  The  life  cycle  of  a  species  of  Crithidia  parasitic  in  the  intestinal 

tract  of  Gerris  fossarum  Fabr.     Ibid.,  12,  131-146,  pi.  9. 

1909.  The  life  cycle  of  a  species  of  Crithidia  parasitic  in  the  intestinal 

tract  of  Tabanus  hilarius  and  Tabanus  spJ     Ibid.,  15,  333-362,  pi. 
30,  2  figs,  in  text. 

PATTON,  W.  S.,  and  CRAGG,  F.  W. 

1913.     A   text   book   of   medical    entomology.      London,    Christian    Literature 

Society  for  India,  pp.   1-768,  pis.   1-89. 
PORTER,  A. 

1909.  The  morphology  and  the  life  cycle  of  Crithidia  gerridis  as  found  in 
the  British  water-bug,  Gerris  paludum.  Parasitology,  2,  348-366, 
pi.  4. 

1909.  The   life   cycle    of   Herpetomonas  jaculum    (Leger)    parasitic   in   the 

alimentary  tract   of  Nepa  cinera.     Ibid.,  2,   367-391,  pi.  5,  1  fig. 
in   text. 

1910.  The    structure    and    life    history    of    Crithidia    melophagia    (Flu)    an 

endoparasite    of   the    sheep-ked,   Melopliagus   ovinus.      Quart.    Jour. 
Micr.  Sci.,  55,  pis.  12-13,  15  figs,  in  text. 

PROWAZEK,  S. 

1904.  Die  Entwicklung  von  Herpetomonas.  Arb.  kais.  Gesundh.,  20,  440- 
452,  7  figs,  in  text. 

S\VEZY,   O. 

1916.  The  kinetonucleus  of  flagellates  and  the  binuclear  theory  of  Hart- 

mann.     Univ.  Calif.  Publ.  Zool.,  16,  185-240,  58  figs,  in  text. 

WENYON,  C.  M. 

1913.  Observations  on  Herpetomonas  muscae  domesticae  and  some  allied 
flagellates  with  special  reference  to  the  structure  of  their  nuclei. 
Arch.  f.  Prot.,  31,  1-36,  pis.  1-3,  6  figs,  and  1  diagram  in  text.  . 


EXPLANATION  OF  PLATES 

All  figures  were  outlined  with  a  camera  lucida  using  a  ^  objective  on  the 
binocular  microscope  and  a  Watson  no.  20  holoscopic  eye-piece.  The  magnifica- 
tion is  in  all  cases  approximately  3500.  Unless  otherwise  stated  all  figures  were 
made  from  iron-haematoxylin  preparations. 

PLATE  2 

Crithidia  euryophthalmi  from  the  ' '  crop ' '  of  Euryophthalmus  convivus. 

Fig.  1.  Small,  oval  infective  spore  which  has  been  casually  ingested  with 
food.  This  spore  shows  the  structure  common  to  these  phases,  thick  periplast, 
deeply  staining  nucleus  in  extreme  posterior  end,  heavily  stained  parabasal 
body.  Faint  nuclear  rhizoplast. 

Figs.  2-8.  A  series  of  developing  crithidias  showing  successive  stages  of 
development.  Both  anterior  and  posterior  ends  are  becoming  attenuate.  The 
nucleus  changes  from  a  solid  mass  of  chromatin  to  a  vesicular  nucleus,  chro- 
matin-encrusted  membrane  and  central  karyosome.  Flagellum  grows  forward 
and  carries  anterior  end  of  body  along  with  it,  which  forms  undulating  membrane. 

Figs.  9-10.  Two  mature  flagellates  illustrating  extremes  in  length,  width, 
and  shape,  common  to  C.  euryophthalmi.  A  whole  series  of  intergrading  forms 
occur  between  these  two  extremes. 

Figs.  11-23.  Multiple  fission;  endogenous  budding.  (Fig.  11).  Flagellate 
with  a  nucleus,  two  endogenous  buds  posterior  to  nucleus.  Chromatin  of  both 
nucleus  and  buds  massed  on  nuclear  membrane.  Blepharoplast  and  parabasal 
body  intact,  no  indications  of  division. 

Fig.  12.  A  short  flagellate;  one  endogenous  bud;  chromatin  peripheral  in 
each  nuclear  structure. 

Fig.  13.  Flagellate  with  three  clearly  defined  nuclear  structures  and  the 
beginning  of  a  third  bud  from  the  most  anterior,  the  nucleus  proper.  Chromatin 
peripheral  in  each  of  these  nuclear  structures. 

Fig.  14.  Elongate  flagellate  nucleus;  two  endogenous  buds  anterior  to  the 
nucleus;  chromatin  peripheral. 

Figs.  15-17.  Pear-shaped  crithidias  with  endogenous  buds.  If  nucleus  be 
present  in  each  there  are  no  differences  in  structure  between  nucleus  and  bud. 
Buds  in  these  flagellates  are  always  sharply  defined.  Chromatin  material  on 
membrane  but  most  of  it  is  in  definite  granules. 

Fig.  18.  Pear-shaped  form  with  numerous  endogenous  buds  but  no  definite 
nucleus.  First  stages  of  degeneration  present,  parabasal  body  has  disappeared 
and  only  a  fragment  of  discarded  flagellum  is  near.  Chromatin  peripheral 
but  also  in  form  of  one  granule. 

Fig.  19.  Elongate  flagellate;  two  endogenous  buds  posterior  to  nucleus. 
Nuclear  rhizoplast  still  present.  Chromatin  distributed  irregularly  on  the 
nuclear  membranes. 

Fig.  20.  Elongate  flagellates  with  nucleus  and  two  endogenous  buds;  chro- 
matin massed  in  the  anterior  and  posterior  portion  of  each  nuclear  structure. 
Parabasal  body  has  taken  no  part  in  this  multiple  fission. 

Fig.  21.  Large  endogenous  flagellate  with  five  buds,  all  anterior  to  nucleus 
proper.  Two  of  the  buds  are  not  destained  sufficiently  to  see  their  structure. 
The  organelles  other  than  nucleus  have  taken  no  part  in  the  process  of  endo- 
genous budding.  Flagellum,  blepharoplast,  parabasal  rhizoplast  and  parabasal 
body  are  still  intact  and  clearly  shown. 

Fig.  22.  Late  stage  in  endogenous  budding.  Buds  have  formed  zooids, 
each  of  which  has  a  nucleus  and  a  second,  deeply-staining  structure  anterior 
to  nucleus.  Flagellum,  parabasal  body,  parabasal  rhizoplast,  and  the  blepharo- 
plast are  still  intact.  No  signs  of  degeneration  are  to  be  observed  in  this 
flagellate. 

Fig.  23.  Drawing  made  from  a  field  literally  covered  by  discarded  flagella 
and  small  zooids.  The  blepharoplast  and  parabasal  body  are  still  attached  to 
8ome  of  the  flagella,  Zooids  show  various  stages  of  nuclear  structure.  Some 
have  a  single  mass  of  chromatin,  others  two  granules  within  a  chromatin-en- 
crusted  membrane.  Some  few  of  the  zooids  contain  a  nucleus,  nuclear  rhizo- 
plast, and  a  second  mass  of  chromatin  anterior  to  these. 

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[McCULLOCH]   PLATE  2 


PLATE  3 

Figs.  24-32.  Multiple  fission;  somatellas.  (Fig.  24).  An  early  stage  in  the 
formation  of  a  somatella.  The  flagellate  is  rounding  up,  and  the  flagellum  is 
entirely  intracellular.  No  indications  of  division  of  the  nucleus,  parabasal 
body,  or  the  blepharoplast  are  present. 

Fig.  25.  A  somewhat  different  type  of  rounding  up.  The  attenuate  anterior 
and  posterior  ends  are  being  wrapped  about  the  body.  The  nucleus  has  begun 
to  constrict  or  to  divide  by  a  process  of  primitive  promitosis.  The  blepharo- 
plast and  parabasal  have  not  yet  begun  to  divide. 

Fig.  26.  A  more  advanced  stage  in  the  formation  of  the  somatella.  The 
flagellum  of  the  rounded  up  flagellate  is  protruding  and  both  the  nucleus  and 
blepharoplast,  together  with  the  parabasal  body,  have  divided.  The  new 
daughter-flagellum  is  not  yet  visible. 

Fig.  27.  A  sphere,  or  somatella,  still  more  advanced  in  its  development. 
Not  only  have  the  blepharoplast,  parabasal  body,  and  the  nucleus  divided  but 
the  new  outgrowth  of  the  second  flagellum  from  the  daughter-blepharoplast  is 
clearly  visible. 

Fig.  28.  A  spherical  crithidia  showing  a  repeated  process  of  division  on 
the  part  of  the  nuclei  and  parabasal  bodies.  Three  parabasal  bodies  and  two 
nuclei  are  present.  There  are  possibly  indications  in  one  of  the  nuclei  wherein 
the  chromatin  has  been  divided  that  another  division  was  about  to  occur. 

Fig.  29.  A  sphere,  or  somatella,  without  protruding  flagella,  containing  at 
least  four  definitely  outlined  merozoites.  The  nuclei,  parabasal  bodies,  nuclear 
rhizoplasts,  and  the  outgrowths  of  the  flagella  are  clearly  visible. 

Fig.  30.  A  densely  stained,  small  somatella  containing  four  relatively  large 
merozoites  which  are  beginning  to  elongate. 

Fig.  31.  A  sphere,  or  somatella,  breaking  up  and  the  merozoites  about  to 
escape.  The  destruction  of  the  sphere  has  occurred  later  than  usual  and  the 
merozoites  have  become  almost  mature  flagellates.  All  nuclear  structures  are 
deeply  stained,  owing  to  the  thickness  of  the  sphere. 

Fig.  32.  An  exceedingly  large  sphere,  comparatively,  showing  many  pro- 
truding flagella.  Here  again  the  nuclear  structures  are  deeply  stained  because 
of  the  thickness  of  the  sphere.  The  exact  number  of  merozoites  cannot  be 
determined,  but  approximately  twenty-four  nuclei  and  parabasal  bodies  can 
be  counted. 

Figs.  33-38.  Binary  fission.  (Fig.  33.)  A  small  spherical  crithidia  under- 
going binary  fission.  The  nucleus  has  divided  but  the  blepharoplast  and  para- 
basal body  show  no  indications  of  fission. 

Fig.  34.  Binary  fission,  in  which  the  blepharoplast  and  parabasal  body  have 
divided  but  the  nucleus  has  not  yet  divided.  A  flagellum  from  the  daughter- 
blepharoplast  has  already  grown  forward. 

Fig.  35.  Binary  fission  taking  place  in  a  developing  crithidia.  Both  the 
nucleus  and  parabasal  body  have  divided,  and  a  new  flagellum  can  be  observed 
growing  from  the  daughter-blepharoplast. 

Fig.  36.  Simple  binary  fission;  blepharoplast,  parabasal  body,  and  nucleus 
have  divided.  The  chromatin  in  the  nuclei  is  peripheral,  about  the  membrane. 

Figs.  37,  38.  A  more  advanced  stage  of  binary  fission,  showing  in  addition 
to  the  division  of  the  blepharoplasts,  parabasal  bodies,  and  nuclei  a  cleavage 
in  the  cytoplasm  to  form  two  crithidias  in  each  case. 

Fig.  39.  Intracellular  multiple  fission:  one  of  the  many  infected  cells 
from  the  "crop"  of  Euryophthalmus  convivus.  This  cell  is  in  a  degenerating 
condition.  The  nucleus  stains  a  blue-gray  color  in  iron-haemotoxylin.  There 
are  at  least  three  and  possibly  five  intracellular  infections  by  C.  euryophthalmi 
in  this  cell.  There  are  approximately  seventy  parasites  within  this  cell:  (a) 
A  group  of  parasites  of  two  sizes,  small  oval  forms,  non-flagellated  and  elon- 
gated Crithidia.  Nuclei  of  all  crithidias  are  diffuse,  possibly  due  to  thickness 
of  smear.  Nucleus  and  parabasal  body  readily  observed  but  the  other  organ- 
elles  are  not  always  clear,  (b)  Another  group  evidently  the  result  of  a  process 
of  intracellular  multiple  fission,  (c)  Similar  to  b.  Circular  cavity  about  these 
non-flagellated  crithidias  may  indicate  the  outline  of  a  former  somatella 
wherein  plasmotomy  has  occurred  early,  (d)  Elongate  merozoites  probably 
resulting  from  another  intracellular  somatella.  Plasmotomy  has  occurred  and 
the  merozoites  are  about  to  make  their  way  out  of  the  host  cell,  (e)  A  scattered 
group  of  oval  merozoites.  Considerable  variation  in  size  is  noted.  Parabasal 
body,  nucleus,  and  intracellular  portions  of  flagellum  clearly  shown.  (/)  A 
mature  merozoite  making  it  way  out  of  the  host  cell.  The  non-flagellated,  or 
posterior,  end  directed  first. 

[184] 


UNIV,   CALIF,   PUBL.  ZOOL   VOL.   19 

^3^ 

V 


[McCULLOCH]   PLATE  3 


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25 


33 


29 


35 


34 


36 


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38 


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-     * 


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*-   * 


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39 


PLATE  4 
Crithidia  euryophthalmi  from  mid-stomach  of  Euryophthalmus  convivus. 

Figs.  40-92.  Nectomonads  (Figs.  40,  41).  Small  oval  forms  at  the  begin- 
ning of  the  developmental  series  of  merozoites  or  zooids  in  the  early  rectal 
phase,  showing  nucleus,  parabasal  body,  nuclear  rhizoplast,  and  light  area  around 
the  parabasal  body.  Nucleus  at  posterior  end  of  the  body.  Anterior  end 
pointed  or  blunt. 

Figs.  42-47.  More  advanced  stages  in  the  developmental  series;  width  of 
body  and  distance  between  nucleus  and  parabasal  are  increasing. 

Figs.  48-54.  Size  of  the  oval  forms  increasing;  nucleus  still  diffuse  and 
located  at  extreme  posterior  end  of  body.  Nuclear  rhizoplast  present,  but  no 
indication  of  the  flagellum  except  in  figure  54. 

Figs.  55-58.  Developing  forms.  Anterior  end  elongating  and  extending 
out  along  the  flagellum.  Nucleus  still  diffuse.  In  figure  58  the  posterior  end 
of  the  body  is  slightly  elongated. 

Fig.  59.  A  group  in  upper  part  of  mid-stomach.  Note  vesicular  type  of 
nucleus,  with  the  centriole-like  karyosome.  Both  ends  of  the  body  are  more 
or  less  pointed.  The  position  of  the  parabasal  body  near  the  nucleus  is  unusual. 

Fig.  60-62.  Both  anterior  and  posterior  ends  are  elongating.  Vesicular 
nu'cleus,  nuclear  rhizoplast,  parabasal  body,  parabasal  rhizoplast,  and  flagellum 
very  distinct. 

Fig.  63.  Posterior  end  blunt;  vesicular  type  of  nucleus.  Parabasal  body 
near  the  nucleus.  Anterior  end  of  the  body  well  developed;  a  slight  undulating 
membrane  present. 

Fig.  64.     Form  a   little   more   developed.     Posterior   end   of   the   body   is 
blunt  or  round,  and  nucleus  is  diffuse. 

Fig.  65.  Almost  mature  flagellate;  nucleus  diffuse;  posterior  end  elongated 
and  quite  broad. 

Fig.  66.  Free  form,  showing  vesicular  nucleus  and  a  distinct  undulating 
membrane. 

Fig.  67.     Nectomonad  developing  into  elongate  flagellate;  nucleus  diffuse. 

Figs.  68-72.  Nectomonads  of  mid-stomach,  long,  slender,  flagellates  show- 
ing the  common  variations  in  the  nuclear  structures.  The  diffuse  type  in 
figure  68;  vesicular  in  figures  69  and  71;  chromatin-encrusted  nuclear  membrane 
in  figures  70  and  72. 


[186] 


UNIV.   CALIF.    PUBL.   ZOOL.    VOL.    19 


[McCULLOCH]   PLATE  4 


» 

40 


*  . 


43       44  45         46 


47 


48 


51 


52 


53 


54 


55 


56 


58 


59 


63 


61 


62 


66 


64 


\ 


65 


69 


70 


PLATE  5 

Figs.  73-92.  Nectomonads  from  pyloric  expansion  (Figs.  73-80).  A  series 
of  elongate,  slender  flagellates  from  crithidial  infection  of  the  pyloric  expan- 
sion. Vesicular  types  of  nuclear  structure  in  figures  73,  75,  80.  Chromatin 
broken  up  into  granules  in  figures  74,  76,  and  79.  Chromatin  of  nucleus  on 
the  membrane  in  figures  77,  78. 

Figs.  81-86.  Another  group  of  nectomonads  from  pyloric  expansion,  illus- 
trating the  changes  which  take  place  in  shape  of  body,  length  decreases,  width 
increases.  These  are  shorter,  broad  types  of  crithidias,  and  probably  come 
from  the  longer,  more  slender  forms.  Nuclear  structure  usually  shows  the 
central  karyosome.  Figure  82  shows  the  chromatin  broken  up,  a  condition  which 
may  indicate  an  early  stage  of  degeneration  of  the  nectomonads. 

Figs.  87-90.  Transition  flagellates  from  nectomonads  to  haptomonads.  By 
a  process  of  binary  fission  these  transition  forms  become  reduced  in  size.  Cer- 
tain nectomonads  become  attached  to  wall  of  the  pyloric  expansion  or  they 
form  haptomonads.  Others  degenerate  before  reaching  this  stage. 

Figs.  91-92.  Free  flagellates.  Their  size  is  doubtless  due  to  a  process  of' 
binary  fission  and  they  resemble  the  attached  haptomonads  of  the  pyloric 
expansion.  They  are  either  nectomonads  which  are  about  to  become  hapto- 
monads or  vice  versa.  If  the  latter,  the  haptomonads  upon  becoming  free  again 
usually  degenerate  at  once. 


[188] 


UNIV.  CALIF.    PUBL.  ZOOL.   VOL.    19 


[McCULLOCHj   PLATE  5 


PLATE  6 

Figs.  93-96.  Haptomonads  from  mid-stomach.  Haptomonads  of  this  type 
may  be  found  attached  to  wall  of  the  ' '  crop, ' '  posterior  part  of  mid-stomach 
or  anterior  part  of  pyloric  expansion.  Small,  slender  flagellates  usually  with 
a  vesicular  type  of  nucleus  are  shown  in  figures  93-95.  Figure  94.  Nuclear 
membrane  distorted,  karyosome  excentric.  Figure  96  may  show  degeneration 
since  there  is  no  nuclear  membrane  and  the  chromatin  is  breaking  up. 

Figs.  97-124.  Haptomonads  from  pyloric  expansion  (Figs.  97-106).  Transi- 
tion haptomonads  lining  mid-portion  of  the  pyloric  expansion.  Bodies  becom- 
ing pear-shaped;  flagella  largely  absorbed  and  intracellular  throughout  length. 
Nucleus  usually  vesicular.  Binary  fission  is  common  among  these  forms.  Their 
size  is  reduced  in  this  way. 

Figs.  107-124.  Small  haptomonads,  found  in  the  extreme  posterior  portions 
of  the  pyloric  expansion.  They  form  a  dense  compact  layer  of  parasites  at- 
tached to  epithelial  lining.  Keduction  in  size  shown,  beginning  with  figure  107. 
Degeneration  is  shown  in  the  nuclear  structure,  as  in  figure  114,  and  in  the 
cytoplasm  of  figures  117,  118.  Figures  118-124  are  all  haptomonads  which  show 
advanced  stages  of  degeneration;  the  cytoplasm  is  vacuolate  and  the  nuclear 
structure  is  no  longer  normal.  Most  of  these  have  become  detached  from  the 
wall  of  the  pyloric  expansion. 

Figs.  125-131.  A  series  of  final  spore  forms  from  rectum,  showing  thick 
periplast,  deeply  stained  cytoplasm,  and  two  chromatin  bodies.  Nuclear  rhizo- 
plast  not  visible  in  these  deeply  stained  spores. 


[190] 


UNIV,   CALIF,    PUBL.   ZOOL.   VOL.    19 


[McCULLOCH]   PLATE  6 


93 


100 


94 


101 


107 


108 


110        115 


IJ7 


118  i\9 


120 


122 


123         124 


125  126 


• 


127 


128 


129  130  131 


UNIVERSITY  OF  CALIFORNIA  PUBLICATIONS— (Continued) 

8.  Osteological  Relationships  of  Three  Species  of  Beavers,  by  F.  Harvey 

Holden.    Pp.  75-114,  platea  6-12,  18  text  figures.    March,  1917 ... .40 

9.  Notes  on  the  Systematic  Status  of  the  Toads  and  Frogs  of  California,  by 

Charles  Lewis  Camp.    Pp.  115-125,  8  text  figures.    February,  1917 .10 

10.  A  Distributional  List  of  the  Amphibians  and  Reptiles  of  California,  by 

Joseph  Grinnell  and  Charles  Lewis  Camp.    Pp.  127-208,  14  figures  in  text. 
July,  1917  _      .85 

11.  A  Study  of  the  Races  of  the  White-Fronted  Goose  (Anser  albifrons)  Occur- 

ring in  California,  by  H.  S.  Swarth  and  Harold  C.  Bryant.    Pp.  209-222, 

2  figures  in  text,  plate  13.    October,  1917 ._„ .15 

12.  A  Synopsis  of  the  Bats  of  California,  by  Hilda  Wood  Grinnell.    Pp.  223-404, 

plates  14-24,  24  text  figures.    January  31,  1918  _ 2.00 

15.  The  Pacific  Coast  Jays  of  the  Genus  Aphelocoma,  by  H.  S.  Swarth.    Pp. 

405-422,  1  figure  in  text.    February  23,  1918 ^. .20 

14.  Six  New  Mammals  from  the  Mohave  Desert  and  Inyo  Regions  of  California, 
by  Joseph  Grinnell.    Pp.  423-430. 

16.  Notes  on  Some  Bats  from  Alaska  and  British  Columbia,  by  Hilda  Wood 

Grinnell.    Pp.  431-433. 

Nos.  14  and  15  in  one  cover.    April,  1918 .15 

16.  Revision  of  the  Rodent  Genus  Aplodontia,  by  Walter  P.  Taylor.    Pp.  435- 

504,  plates  25-29,  16  text  figures.    May,  1918 75 

17.  The  Subspecies  of  the  Mountain  Chickadee,  by  Joseph  Grinnell.    Pp.  605- 

515,  3  text  figures.    May,  1918  15 

18.  Excavations  of  Burrows  of  the  Rodent  Aplodontia,  with  Observations  on 

the  Habits  of  the  Animal,  by  Charles  Lewis  Camp.    Pp.  517-536,  6  figures 

in  text.    June,  1918  .20 

Index,  pp.  537-545. 

VoL  18.    1.  Mitosis  in  Giardia  microti,  by  William  C.  Boeck.     Pp.  1-26,  plate  1.    Octo- 
ber   1917  .35 

***'*>      •* •*'•*  *       .......-.«——.—•-...••-.•.*.»••. 

2.  An  Unusual  Extension  of  the  Distribution  of  the  Shipworm  in  San  Fran- 
cisco Bay,  California,  by  Albert  L.  Barrows.  Pp.  27-43.  December,  1917.  .20 

8.  Description  of  Some  New  Species  of  Polynoidae  from  the  Coast  of  Cali- 
fornia, by  Christine  Essenberg.  Pp.  45-60,  plates  23.  October,  1917  —  .20 

4.  New  Species  of  AmpMnomidae  from  the  Pacific  Coast,  by  Christine  Essen- 

berg.    Pp.  61-74,  plates  4-5.    October,  1917  — 15 

5.  Crithidia  euryopMhalmi,  sp.  nov.,  from  the  Hemipteran  Bug,  Euryophthalmus 

convivus  Stal,  by  Irene  McCulloch.    Pp.  75-88,  35  text  figures.    Decem- 
ber, 1917  .15 

6.  On  the  Orientation  of  Erythropsis,  by  Charles  Atwood  Kofoid  and  Olive 

Swezy.  Pp.  89-102,  12  figures  in  text.    December,  1917 15 

7.  The  Transmission  of  Nervous  Impulses  in  Relation  to  Locomotion  in  the 

Earthworm,  by  John  F.  Bovard.    Pp.  103-134, 14  figures  in  text.    January, 
1918    „ „ .35 

8.  The  Function  of  the  Giant  Fibers  in  Earthworms,  by  John  F.  Bovard.    Pp. 

135-144,  1  figure  in  text.    January,  1918 - ._ —      .10 

9.  A  Rapid  Method  for  the  Detection  of  Protozoan  Cysts  in  Mammalian 

Faeces,  by  William  C.  Boeck.    Pp.  145-149.    December,  1917 ~      .05 

10.  The  Musculature  of  Heptanchus  maculatus,  by  Pirie  Davidson...  Pp.  151-170, 

12  figures  in  text.    March,  1918 _ — 25 

11.  The  Factors  Controlling  the  Distribution  of  the  Polynoidae  of  the  Pacific 

Coast  of  North  America,  by  Christine  Essenberg.    Pp.  171-238,  plates  6-8, 

2  figures  in  text.    March,  1918-. ~ —      .75 

12.  Differentials  in  Behavior  of  the  Two  Generations  of  Salpa  dvmocratica 

Relative  to  the  Temperature  of  the  Sea,  by  Ellis  L.  MichaeL    Pp.  239-298, 
plates  9-11, 1  figure  in  text.    March,  1918  — 

13.  A  Quantitative  Analysis  of  the  Molluscan  Fauna  of  San  Francisco  Bay,  by 

E.  L.  Packard.    Pp.  299-336,  plates  12-13,  6  figs,  in  text.    April,  1918 —      .40 

14.  The  Neuromotor  Apparatus  of  Euplotes  patella,  by  Harry  B.  Yocom.    Pp. 

337-396,  plates  14-16.    September,  1918  _ .70 

15.  The  Significance  of  Skeletal  Variations  in  the  Genus  Peridinium,  by  A.  L. 

Barrows.    Pp.  397-478,  plates  17-20,  19  figures  in  text.    June,  1918 90 


UNIVERSITY  OF  CALIFORNIA  PUBLICATIONS— (Continued) 

16.  The  Subclavian  Vein  and  its  Relations  in  Elasmobranch  Fishes,  by  J. 

Frank  DanieL    Pp.  479-484,  2  figures  in  text.    August,  1918 .10 

17.  The  Cercaria  of  the  Japanese  Blood  Fluke,  Schistosoma  japonicum  Kat- 

surada,  by  William  W.  Cort.    Pp.  485-507,  3  figures  in  text. 

18.  Notes  on  the  Eggs  and  Miracidia  of  the  Human  Schistosomes,  by  William 

W.  Cort.    Pp.  509-519,  7  figures  in  text. 

Nos.  17  and  18  In  one  cover.    January,  1919 _ 35 

Index  in  preparation. 

Vol.19.  1.  Reaction  of  Various  Plankton  Animals  with  Reference  to  their  Diurnal 

Migrations,  by  Calvin  O.  Esterly.    Pp.  1-83.    April,  1919 85 

2.  The  Pteropod  Desmopterus  pacificus   (sp.  nov.),  by  Christine  Essenberg. 

Pp.  85-88,  2  figures  in  text.     May,  1919  05 

3.  Studies  on  Giardia  microti,  by  William  0.  Boeck.     Pp.  85-136,  plate  1,  19 

figures  in  text  60 

4.  A  Comparison  of  the  Life  Cycle  of  Crithidia  With  that  of  Trypanosoma  in 

the  Invertebrate  Host,  by  Irene  McCulloch.  Pp.  135-190,  plates  2-6,  3 
figures  in  text.  October,  1919  60 

5.  A  Muscid  Larva  of  the  San  Francisco  Bay  Region  which  Sucks  the  Blood 

of  Nestling  Birds,  by  O.  E.  Plath.  Pp.  191-200.    February,  1919 .10 

6.  Binary  Fission  in  Collodictyon  triciUatum  Carter,  by  Robert  Clinton  Rhodes. 

Pp.  201-274,  plates  7-14,  4  figures  in  text  (In  press) 

7.  The  Excretory  System  of  a  Stylet  Cercaria,  by  William  W.  Cort.    Pp.  275- 

281,  1  figure  in  text.    August,  1919 10 

Vol.20.  1.  Studies  on  the  Parasites  of  the  Termites  I.  On  Streblomastix  strix,  a 
Polymastigote  Flagellate  with  a  Linear  Plasmodial  Phase,  by  Charles 
Atwood  Kofoid  and  Olive  Swezy.  Pp.  1-20,  plates  1-2,  1  figure  in 
text.  July,  1919 25 

2.  Studies  on  the  Parasites  of  the  Termites  II.     On  Trichomitus  termitidis, 

a  Polymastigote  Flagellate  with  a  Highly  Developed  Neuromotor  System, 
by  Charles  Atwood  Kofoid  and  Olive  Swezy.  Pp.  21-40,  plates  3-4,  2 
figures  in  text.  July,  1919  - .25 

3.  Studies  on  the  Parasites  of  the  Termites  III.    On  Trichonymplia  campanula 

Sp.  Nov.,  by  Charles  Atwood  Kofoid  and  Olive  Swezy.  Pp.  41-98,  plates 
5-12,  4  figures  in  text.  July,  1919  :. 75 

4.  Studies  on  the  Parasites  of  the  Termites  IV.     On  Leidyopsis  sphaerica 

Gen.  Nov.,  Sp.  Nov.,  by  Charles  Atwood  Kofoid  and  Olive  Swezy.  Pp. 
99-116,  plates  13-14,  1  figure  in  text.  July,  1919.._ „ .25 

Vol.  21.  1.  A  Revision  of  the  Microtus  calif  ornicus  Group  of  Meadow  Mice,  by  Rem- 
ington Kellogg.  Pp.  1-42,  1  figure  in  text.  December,  1918 50 

2.  Five  New  Five-toed  Kangaroo  Rats  from  California,  by  Joseph  Grinnell. 

Pp.  43-47.    March,  1919  05 


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